CTLA4/CD28 ligands and uses therefor

ABSTRACT

Nucleic acids encoding novel CTLA4/CD28 ligands which costimulate T cell activation are disclosed. In one embodiment, the nucleic acid has a sequence which encodes a B lymphocyte antigen, B7-2. Preferably, the nucleic acid is a DNA molecule comprising at least a portion of a nucleotide sequence shown in FIG. 8, SEQ ID NO:1 or FIG. 14, SEQ ID NO:23. The nucleic acid sequences of the invention can be integrated into various expression vectors, which in turn direct the synthesis of the corresponding proteins or peptides in a variety of hosts, particularly eukaryotic cells, such as mammalian and insect cell culture. Also disclosed are host cells transformed to produce proteins or peptides encoded by the nucleic acid sequences of the invention and isolated proteins and peptides which comprise at least a portion of a novel B lymphocyte antigen. Proteins and peptides described herein can be administered to subjects to enhance or suppress T cell-mediated immune responses.

GOVERNMENT FUNDING

Work described herein was supported under CA-40216-08 awarded by theNational Institutes of Health. The U.S. government therefore may havecertain rights in this invention.

RELATED APPLICATIONS

This application is a continution-in-part of U.S. Ser. No. 08/280,757,entitled "Novel CTLA4/CD28 Ligands and Uses Therefor" filed Jul. 26,1994, which is a continuation-in-part of U.S. Ser. No. 08/109,393,entitled "Novel CTLA4/CD28 Ligands and Uses Therefor" filed Aug. 19,1993, now abandoned, which is a continuation-in-part of U.S. Ser. No.08/101,624, also entitled "Novel CTLA4/CD28 Ligands and Uses Therefor",filed Jul. 26, 1993. This application is also a continution-in-part ofU.S. Ser. No. 08/147,773, entitled "Tumor Cells Modified To Express B7-2And B7-3 With Increased Immunogenicity And Uses Therefor" filed Nov. 3,1993 now abandoned. The contents of each of these applications isincorporated herein by reference.

BACKGROUND OF THE INVENTION

To induce antigen-specific T cell activation and clonal expansion, twosignals provided by antigen-presenting cells (APCs) must be delivered tothe surface of resting T lymphocytes (Jenkins, M. and Schwartz, R.(1987) J. Exp. Med. 165, 302-319; Mueller, D. L., et al. (1990) J.Immunol. 144, 3701-3709; Williams, I. R. and Unanue, E. R. (1990) J.Immunol. 145, 85-93). The first signal, which confers specificity to theimmune response, is mediated via the T cell receptor (TCR) followingrecognition of foreign antigenic peptide presented in the context of themajor histocompatibility complex (MHC). The second signal, termedcostimulation, induces T cells to proliferate and become functional(Schwartz, R. H. (1990) Science 248, 1349-1356). Costimulation isneither antigen-specific, nor MHC restricted and is thought to beprovided by one or more distinct cell surface molecules expressed byAPCs (Jenkins, M. K., et al. (1988) J. Immunol. 140, 3324-3330; Linsley,P. S., et al. (1991) J. Exp. Med. 173, 721-730; Gimmi, C. D., et al.,(1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Young, J. W., et al.(1992) J. Clin. Invest. 90, 229-237; Koulova, L., et al. (1991) J. Exp.Med. 173, 759-762; Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA.89, 271-275; van-Seventer, G. A., et al. (1990) J. Immunol. 144,4579-4586; LaSalle, J. M., et al., (1991) J. Immunol. 147, 774-80;Dustin, M. I., et al., (1989) J. Exp. Med. 169, 503; Armitage, R. J., etal. (1992) Nature 357, 80-82; Liu, Y., et al. (1992) J. Exp. Med. 175,437-445).

Considerable evidence suggests that the B7 protein, expressed on APCs,is one such critical costimulatory molecule (Linsley, P. S., et al.,(1991) J. Exp. Med. 173, 721-730; Gimmi, C. D., et al., (1991) Proc.Natl. Acad. Sci. USA. 8, 6575-6579; Koulova, L., et al., (1991) J. Exp.Med. 173, 759-762; Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA.89, 271-275; Linsley, P. S. et al. (1990) Proc. Natl. Acad. Sci. USA.87, 5031-5035; Freeman, G. J. et al. (1991) J. Exp. Med. 174,625-631.).B7 is the counter-receptor for two ligands expressed on T lymphocytes.The first ligand, termed CD28, is constitutively expressed on resting Tcells and increases after activation. After signaling through the T cellreceptor, ligation of CD28 induces T cells to proliferate and secreteIL-2 (Linsley, P. S., et al. (1991) J. Exp. Med. 173, 721-730; Gimmi, C.D., et al. (1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Thompson,C. B., et al. (1989) Proc. Natl. Acad. Sci. USA. 86, 1333-1337; June, C.H., et al. (1990) Immunol. Today. 11, 211-6; Harding, F. A., et al.(1992) Nature. 356, 607-609.). The second ligand, termed CTLA4 ishomologous to CD28 but is not expressed on resting T cells and appearsfollowing T cell activation (Brunet, J. F., et al., (1987) Nature 328,267-270). DNA sequences encoding the human and murine CTLA4 protein aredescribed in Dariavich, et al. (1988) Eur. J. Immunol. 18(12),1901-1905; Brunet, J. F., et al. (1987) supra; Brunet, J. F. et al.(1988) Immunol. Rev. 103:21-36; and Freeman, G. J., et al. (1992) J.Immunol. 149, 3795-3801. Although B7 has a higher affinity for CTLA4than for CD28 (Linsley, P. S., et al., (1991) J. Exp. Med. 174,561-569), the function of CTLA4 is still unknown.

The importance of the B7:CD28/CTLA4 costimulatory pathway has beendemonstrated in vitro and in several in vivo model systems. Blockade ofthis costimulatory pathway results in the development of antigenspecific tolerance in murine and humans systems (Harding, F. A., et al.(1992) Nature. 356, 607-609; Lenschow, D. J., et al. (1992) Science.257, 789-792; Turka, L. A., et al. (1992) Proc. Natl. Acad. Sci. USA.89, 11102-11105; Gimmi, C. D., et al. (1993) Proc. Natl. Acad. Sci USA90, 6586-6590; Boussiotis, V., et al. (1993) J. Exp. Med. 178,1753-1763). Conversely, expression of B7 by B7 negative murine tumorcells induces T-cell mediated specific immunity accompanied by tumorrejection and long lasting protection to tumor challenge (Chen, L., etal. (1992) Cell 71, 1093-1102; Townsend, S. E. and Allison, J. P. (1993)Science 259, 368-370; Baskar, S., et al. (1993) Proc. Natl. Acad. Sci.90, 5687-5690.). Therefore, manipulation of the B7:CD28/CTLA4 pathwayoffers great potential to stimulate or suppress immune responses inhumans.

SUMMARY OF THE INVENTION

This invention pertains to isolated nucleic acids encoding novelmolecules which costimulate T cell activation. Preferred costimulatorymolecules include antigens on the surface of B lymphocytes, professionalantigen presenting cells (e.g., monocyes cells, Langerhan cells) andother cells (e.g., keratinocytes, endothelial cells, astrocytes,fibroblasts, oligodendrocytes) which present antigen to immune cells,and which bind either CTLA4, CD28, both CTLA4 and CD28 or other known oras yet undefined receptors on immune cells. Such costimulatory moleculesare referred to herein as CTLA4/CD28 binding counter-receptors or Blymphocyte antigens, and are capable of providing costimulation toactivated T cells to thereby induce T cell proliferation and/or cytokinesecretion. Preferred B lymphocyte antigens include B7-2 and B7-3 andsoluble fragments or derivatives thereof which bind CTLA4 and/or CD28and have the ability to inhibit or induce costimulation of immune cells.In one embodiment, an isolated nucleic acid which encodes a peptidehaving the activity of the human B7-2 B lymphocyte antigen is provided.Preferably, the nucleic acid is a cDNA molecule having a nucleotidesequence encoding human B7-2, as shown in FIG. 8 (SEQ ID NO:1). Inanother embodiment, the nucleic acid is a cDNA molecule having anucleotide sequence encoding murine B7-2, as shown in FIG. 14 (SEQ IDNO:22).

The invention also features nucleic acids which encode a peptide havingB7-2 activity and at least about 50%, more preferably at least about 60%and most preferably at least about 70% homologous with an amino acidsequence shown in FIG. 8 (SEQ ID NO:2) or an amino acid sequence shownin FIG. 14 (SEQ ID NO:23). Nucleic acids which encode peptides havingB7-2 activity and at least about 80%, more preferably at least about90%, more preferably at least about 95% and most preferably at leastabout 98% or at least about 99% homologous with an amino acid sequenceshown in FIG. 8 (SEQ ID NO:2) or an amino acid sequence shown in FIG. 14(SEQ ID NO:23) are also within the scope of the invention. In anotherembodiment, the peptide having B7-2 activity is encoded by a nucleicacid which hybridizes under high or low stringency conditions to anucleic acid which encodes a peptide having an amino acid sequence ofFIG. 8 (SEQ ID NO:2) or a peptide having an amino acid sequence shown inFIG. 14 (SEQ ID NO:23).

The invention further pertains to an isolated nucleic acid comprising anucleotide sequence encoding a peptide having B7-2 activity and having alength of at least 20 amino acid residues. Peptides having B7-2 activityand consisting of at least 40 amino acid residues in length, at least 60amino acid residues in length, at least 80 amino acid residues inlength, at least 100 amino acid residues in length or at least 200 ormore amino acid residues in length are also within the scope of thisinvention. Particularly preferred nucleic acids encode a peptide havingB7-2 activity, a length of at least 20 amino acid residues or more andat least 50% or greater homology (preferably at least 70%) with asequence shown in FIG. 8 (SEQ ID NO:2).

In one preferred embodiment, the invention features an isolated DNAencoding a peptide having B7-2 activity and an amino acid sequencerepresented by a formula:

    X.sub.n --Y--Z.sub.m

In the formula, Y consists essentially of amino acid residues 24-245 ofthe sequence shown in FIG. 8 (SEQ ID NO:2). X_(n) and Z_(m) areadditional amino acid residue(s) linked to Y by an amide bond. X_(n) andZ_(m) are amino acid residues selected from amino acid residuescontiguous to Y in the amino acid sequence shown in FIG. 8 (SEQ IDNO:2). X_(n) is amino acid residue(s) selected from amino acidscontiguous to the amino terminus of Y in the sequence shown in FIG. 8(SEQ ID NO:2), i.e., selected from amino acid residue 23 to 1. Z_(m) isamino acid residue(s) selected from amino acids contiguous to thecarboxy terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO:2),i.e., selected from amino acid residue 246 to 329. According to theformula, n is a number from 0 to 23 (n=0-23) and m is a number from 0 to84 (m=0-84). A particularly preferred DNA encodes a peptide having anamino acid sequence represented by the formula X_(n) --Y--Z_(m), where Yis amino acid residues 24-245 of the sequence shown in FIG. 8 (SEQ IDNO:2) and n=0 and m=0.

The invention also features an isolated DNA encoding a B7-2 fusionprotein which includes a nucleotide sequence encoding a first peptidehaving B7-2 activity and a nucleotide sequence encoding a second peptidecorresponding to a moiety that alters the solubility, binding affinity,stability or valency of the first peptide. Preferably, the first peptidehaving B7-2 activity includes an extracellular domain portion of theB7-2 protein (e.g., about amino acid residues 24-245 of the sequenceshown in FIG. 8 (SEQ ID NO:2)) and the second peptide is animmunoglobulin constant region, for example, a human Cγ1 or Cγ4 domain,including the hinge, CH2 and CH3 region, to produce a B7-2immunoglobulin fusion protein (B7-2Ig)(see Capon et al. (1989) Nature337, 525-531 and Capon U.S. Pat. No. 5,116,964).

The nucleic acids obtained in accordance with the present invention canbe inserted into various expression vectors, which in turn direct thesynthesis of the corresponding protein or peptides in a variety ofhosts, particularly eucaryotic cells, such as mammalian and insect cellculture, and procaryotic cells such as E. coli. Expression vectorswithin the scope of the invention comprise a nucleic acid encoding atleast one peptide having the activity of a novel B lymphocyte antigen asdescribed herein, and a promoter operably linked to the nucleic acidsequence. In one embodiment, the expression vector contains a DNAencoding a peptide having the activity of the B7-2 antigen and a DNAencoding a peptide having the activity of another B lymphocyte antigen,such as the previously characterized B7 activation antigen, referred toherein as B7-1. Such expression vectors can be used to transfect hostcells to thereby produce proteins and peptides, including fusionproteins, encoded by nucleic acids as described herein.

Nucleic acid probes useful for assaying a biological sample for thepresence of B cells expressing the B lymphocyte antigens B7-2 and B7-3are also within the scope of the invention.

The invention further pertains to isolated peptides having the activityof a novel B lymphocyte antigen, including the B7-2 and B7-3 proteinantigens. A preferred peptide having B7-2 activity is produced byrecombinant expression and comprises an amino acid sequence shown inFIG. 8 (SEQ ID NO:2). Another preferred peptide having B7-2 activitycomprises an amino acid sequence shown in FIG. 14 (SEQ ID NO:23). Aparticularly preferred peptide having the activity of the B7-2 antigenincludes at least a portion of the mature form of the protein, such asan extracellular domain portion (e.g., about amino acid residues 24-245of SEQ ID NO:2) which can be used to enhance or suppress T-cell mediatedimmune responses in a subject. Other preferred peptides having B7-2activity include peptides having an amino acid sequence represented by aformula:

    X.sub.n --Y--Z.sub.m

In the formula, Y is amino acid residues selected from the groupconsisting of: amino acid residues 55-68 of the sequence shown in FIG. 8(SEQ ID NO:2); amino acid residues 81-89 of the sequence shown in FIG. 8(SEQ ID NO:2); amino acid residues 128-142 of the sequence shown in FIG.8 (SEQ ID NO:2); amino acid residues 160-169 of the sequence shown inFIG. 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shownin FIG. 8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequenceshown in FIG. 8 (SEQ ID NO:2). In the formula X_(n) and Z_(m) areadditional amino acid residue(s) linked to Y by an amide bond and areselected from amino acid residues contiguous to Y in the amino acidsequence shown in FIG. 8 (SEQ ID NO:2). X_(n) is amino acid residue(s)selected from amino acids contiguous to the amino terminus of Y in thesequence shown in FIG. 8 (SEQ ID NO:2). Z_(m) is amino acid residue(s)selected from amino acids contiguous to the carboxy terminus of Y in thesequence shown in FIG. 8 (SEQ ID NO:2). According to the formula, n is anumber from 0 to 30 (n=0-30) and m is a number from 0 to 30 (m=0-30).

Fusion proteins or hybrid fusion proteins including a peptide having theactivity of a novel B lymphocyte antigen (e.g., B7-2, B7-3) are alsofeatured. For example, a fusion protein comprising a first peptide whichincludes an extracellular domain portion of a novel B lymphocyte antigenfused to second peptide, such as an immunoglobulin constant region, thatalters the solubility, binding affinity, stability and/or valency of thefirst peptide are provided. In one embodiment, a fusion protein isproduced comprising a first peptide which includes amino acid residuesof an extracellular domain portion of the B7-2 protein joined to asecond pepide which includes amino acid residues of a sequencecorresponding to the hinge, CH2 and CH3 regions of Cγ1 or Cγ4 to form aB7-2Ig fusion protein. In another embodiment, a hybrid fusion protein isproduced comprising a first peptide which includes an extracellulardomain portion of the B7-1 antigen and an extracellular domain portionof the B7-2 antigen and a second peptide which includes amino acidresidues corresponding to the hinge, CH2 and CH3 of C71 (see e.g.,Linsley et al. (1991) J. Exp. Med. 1783:721-730; Capon et al. (1989)Nature 33, 525-53 1; and Capon U.S. Pat. No. 5,116,964). In a yetanother embodiment, a hybrid fusion protein comprises theimmunoglobulin-like variable domain of B7-2, but not theimmunoglobulin-like constant domain of B7-2, linked to the constantregion of an immunoglobulin molecule. In a preferred embodiment, theB7-2Ig fusion protein includes the variable domain of human B7-2,preferably from about amino acid residue 24 to about amino acid residue133 of the human B7-2 protein (as shown SEQ ID NO:2), fused to theconstant region of an IgG molecule.

Isolated peptides and fusion proteins of the invention can beadministered to a subject to either upregulate or inhibit the expressionof one or more B lymphocyte antigens or block the ligation of one ormore B lymphocyte antigens to their natural ligand on immune cells, suchas T cells, to thereby provide enhancement or suppression ofcell-mediated immune responses in vivo.

Another embodiment of the invention provides antibodies, preferablymonoclonal antibodies, specifically reactive with a peptide of a novel Blymphocyte antigen or fusion protein as described herein. Preferredantibodies are anti-human B7-2 monoclonal antibodies produced byhybridoma cells HF2.3D1, HA5.2B7 and HA3.1F9. These hybridoma cells havebeen deposited with the American Type Culture Collection at ATCCAccession No. HB11686 (HF2.3D1), ATCC Accession No. HB 11687 (HA5.2B7),and ATCC Accession No. HB11688 (HA3.1F9).

A still further aspect of the invention involves the use of the nucleicacids of the invention, especially the cDNAs, to enhance theimmunogenicity of a mammalian cell. In preferred embodiments, themammalian cell is a tumor cell, such as a sarcoma, a lymphoma, amelanoma, a neuroblastoma, a leukemia or a carcinoma, or an antigenpresenting cell, such as a macrophage, which is transfected to allowexpression of a peptide having the activity of a novel B lymphocyteantigen of the invention on the surface of the cell. Macrophages thatexpress a peptide having the activity of a B lymphocyte antigen, such asthe B7-2 antigen, can be used as antigen presenting cells, which, whenpulsed with an appropriate pathogen-related antigen or tumor antigen,enhance T cell activation and immune stimulation.

Mammalian cells can be transfected with a suitable expression vectorcontaining a nucleic acid encoding a peptide having the activity of anovel B lymphocyte antigen, such as the B7-2 antigen, ex vivo and thenintroduced into the host mammal, or alternatively, cells can betransfected with the gene in vivo via gene therapy techniques. Forexample, the nucleic acid encoding a peptide having B7-2 activity can betransfected alone, or in combination with nucleic acids encoding othercostimulatory molecules. In enhancing the immunogenicity of tumors whichdo not express Class I or Class II MHC molecules, it may be beneficialto additionally transfect appropriate class I or II genes into themammalian cells to be transfected with a nucleic acid encoding a peptidehaving the activity of a B lymphocyte antigen, as described herein.

The invention also provides methods for inducing both generalimmunosuppression and antigen-specific tolerance in a subject by, forexample, blocking the functional interaction of the novel B lymphocyteantigens of the invention, e.g., B7-2 and B7-3, to their naturalligand(s) on T cells or other immune system cells, to thereby blockco-stimulation through the receptor-ligand pair. In one embodiment,inhibitory molecules that can be used to block the interaction of thenatural human B7-2 antigen to its natural ligands (e.g., CTLA4 and CD28)include a soluble peptide having B7-2 binding activity but lacking theability to costimulate immune cells, antibodies that block the bindingof B7-2 to its ligands and fail to deliver a co-stimulatory signal (socalled "blocking antibodies", such as blocking anti-B7-2 antibodies),B7-2-Ig fusion proteins, which can be produced in accordance with theteachings of the present invention, as well as soluble forms of B7-2receptors, such as CTLA4Ig or CD28Ig. Such blocking agents can be usedalone or in combination with agents which block interaction of othercostimulatory molecules with their natural ligands (e.g., anti-B7antibody). Inhibition of T cell responses and induction of T celltolerance according to the methods described herein may be usefulprophylactically, in preventing transplantation rejection (solid organ,skin and bone marrow) and graft versus host disease, especially inallogeneic bone marrow transplantation. The methods of the invention mayalso be useful therapeutically, in the treatment of autoimmune diseases,allergy and allergic reactions, transplantation rejection, andestablished graft versus host disease in a subject.

Another aspect of the invention features methods for upregulating immuneresponses by delivery of a costimulatory signal to T cells through useof a stimulatory form of B7-2 antigen, which include soluble,multivalent forms of B7-2 protein, such as a peptide having B7-2activity and B7-2 fusion proteins. Delivery of a stimulatory form ofB7-2 in conjunction with antigen may be useful prophylactically toenhance the efficacy of vaccination against a variety of pathogens andmay also be useful therapeutically to upregulate an immune responseagainst a particular pathogen during an infection or against a tumor ina tumor-bearing host.

The invention also features methods of identifying molecules which caninhibit either the interaction of B lymphocyte antigens, e.g., B7-2,B7-3, with their receptors or interfere with intracellular signallingthrough their receptors. Methods for identifying molecules which canmodulate the expression of B lymphocyte antigens on cells are alsoprovided. In addition, methods for identifying cytokines produced inresponse to costimulation of T cells by novel B lymphocyte antigens arewithin the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are graphic representations of the responses of CD28⁺ Tcells, as assessed by ³ H-thymidine incorporation or IL-2 secretion, tocostimulation provided by either B7 (B7-1) transfected CHO cells (panela) or syngeneic activated B lymphocytes (panel b) cultured in media,anti-CD3 alone, or anti-CD3 in the presence of the following monoclonalantibodies or recombinant proteins: αB7 (133, anti-B7-1); CTLA4Ig; Fab αCD28; control Ig fusion protein (isotype control for CTLA4Ig); or αBγ(anti-B5, the isotype control for anti-B7-1).

FIGS. 2A-2C are graphs of log fluorescence intensity of cell surfaceexpression of B7-1 on splenic B cells activated with surfaceimmunoglobulin (sIg) crosslinking. The total (panel a), B7-1 positive(B7-1⁺, panel b) and B7-1 negative (B7-1⁻, panel c) activated B cellswere stained with anti-B7-1 monoclonal antibody (133) and fluorosceinisothiocyanate (FITC) labeled goat anti-mouse immunoglobulin andanalyzed by flow cytometry.

FIGS. 3A-3B are graphic representations of the responses of CD28⁺ Tcells, as assessed by ³ H-thymidine incorporation and IL-2 secretion, tocostimulation provided by B7-1⁺ (panel a) or B7-1⁻ (panel b) activatedsyngeneic B lymphocytes cultured in media, anti-CD3 alone, or anti-CD3in the presence of the following monoclonal antibodies or recombinantproteins: αBB-1 (133, anti-B7-1 and anti-B7-3); αB7 (anti-B7-1);CTLA4Ig; Fab αCD28; control Ig fusion protein or αB5 (anti-B5).

FIG. 4 is a graphic representation of the cell surface expression ofB7-1, B7-3 and total CTLA4 counter-receptors on fractionated B7-1⁺ andB7-1⁻ activated B lymphocytes.

FIG. 5 is a graphic representation of temporal surface expression ofB7-1 (CTLA4Ig and mAbs BB-1 and 133), B7-3 (CTLA4Ig and mAb BB1) andB7-2 (CTLA4Ig) counter-receptors on splenic B cells activated by sIgcrosslinking.

FIG. 6 is a graphic representation of temporal surface expression ofB7-1 (CTLA4Ig and mAbs BB-1 and 133), B7-3 (CTLA4Ig and mAb BB1) andB7-2 (CTLA4Ig) counter-receptors on splenic B cells activated by MHCclass II crosslinking.

FIGS. 7A-7B are graphic representations of the response of CD28⁺ Tcells, as assessed by ³ H-thymidine incorporation and IL-2 secretion, tocostimulation provided by syngeneic B lymphocytes activated by sIgcrosslinking for 24 hours (panel a) or 48 hours (panel b) and culturedin media, anti-CD3 alone, or anti-CD3 in the presence of the followingmonoclonal antibodies or recombinant protein: αB7(133, anti-B7-1); αBB1(anti-B7-1, anti-B7-3) CTLA4Ig; Fab αCD28; and αB5(anti-B5).

FIGS. 8A-8B are the nucleotide and deduced amino acid sequence of thehuman B lymphocyte antigen B7-2 (hB7-2-clone29).

FIG. 9 is a graphic representation of COS cells transfected with controlplasmid (pCDNAI), plasmid expressing B7-1 (B7-1), or plasmid expressingB7-2 (B7-2) stained with either control mAb (IgM), anti-B7-1 (mAbs 133and BB-1), recombinant protein CTLA4Ig, or isotype matched control Igprotein followed by the appropriate second FITC labelled immunoglobulinand analyzed by flow cytometry.

FIGS. 10A-10B show RNA blot analyses of B7-2 expression in unstimulatedand anti-Ig activated human spenic B cells and cell lines (panel a) andhuman myelomas (panel b).

FIGS. 11A-11B are a graphic representation of the proliferation of CD28+T cells, as assessed by ³ H-thymidine incorporation or IL-2 secretion,to submitogenic stimulation with phorbol myristic acid (PMA) and COScells transfected with vector alone or vectors directing the expressionof either B7-1 or B7-2.

FIGS. 12A-12G are a graphic representation of the inhibition by mAbs andrecombinant proteins of the proliferation of CD28+ T cells, as assessedby ³ H-thymidine incorporation and IL-2 secretion, to stimulation by PMAand COS cells transfected with vector alone (vector), or with a vectorexpressing B7-1 (B7-1) or B7-2 (B7-2). Inhibition studies were performedwith the addition of either no antibody (no mAb), anti-B7 mAb 133 (133),anti-B7 mAb BB-1 (BB1), anti-B5 mAb (B5), Fab fragment of anti-CD28(CD28 Fab), CTLA4Ig (CTLA4Ig), or Ig control protein (control Ig) to thePMA stimulated COS cell admixed CD28⁺ T cells.

FIGS. 13A-13B show the sequence homology between the human B7-2 protein(h B7-2) deduced amino acid sequence (SEQ ID NO:2) and the amino acidsequence of both the human B7-1 protein (h B7-1) (SEQ ID NO:28 and 29)and the murine B7-1 protein (m B7) (SEQ ID NO:30 and 31).

FIGS. 14A-14D are the nucleotide and deduced amino acid sequence of themurine B7-2 antigen (mB7-2) (SEQ ID NO:22 and 23).

FIG. 15 is a graphic representation of the competitive inhibition ofbinding of biotinylated-CTLA4Ig to immobilized B7-2 Ig by B7 family-Igfusion proteins. The Ig fusion proteins examined as competitors were:full-length B7-2 (hB7.2), full-length B7-1 (hB7.1), the variableregion-like domain of B7-2 (hB7.2V) or the constant region-like domainof B7-2 (hB7.2C).

FIGS. 16A-16B are graphic representations of the competitive inhibitionof binding of biotinylated-B7-1-Ig (panel A) or B7-2-Ig (panel B) toimmobilized CTLA4-Ig by increasing concentrations of unlabelled B7-1-Ig(panel A) or B7-2-Ig (panel B). The experimentally determined IC₅₀values are indicated in the upper right corner of the panels.

FIGS. 17A-17C depicts flow cytometric profiles of cells stained with ananti-hB7-2 monoclonal antibody, HA3.1F9. Cells stained with the antibodywere CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH 3T3cells transfected to express human B7-2 (3T3-hB7.2) and controltransfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70 wasused as a positive control.

FIGS. 18A-18C depict flow cytometric profiles of cells stained with ananti-hB7-2 monoclonal antibody, HA5.2B7. Cells stained with the antibodywere CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH 3T3cells transfected to express human B7-2 (3T3-hB7.2) and controltransfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70 wasused as a positive control.

FIGS. 19A-19C depict flow cytometric profiles of cells stained with ananti-hB7-2 monoclonal antibody, HF2.3D1. Cells stained with the antibodywere CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH 3T3cells transfected to express human B7-2 (3T3-hB7.2) and controltransfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70 wasused as a positive control.

FIG. 20 is a graphic representation of the direct binding of solublebiotinylated CTLA4Ig to B7-1Ig, B7-1VIg, B7-1CIg, B7-2Ig, B7-2VIg,B7-2CIg, or human IgG (hIgG) bound to plates.

FIGS. 21A-21E depict flow cytometric profiles of binding of B7-2Ig(Panel C), B7-2VIg (Panel D), B7-1Ig (Panel E), or secondary antibodyalone (Panel B) to CTLA4+ CHO cells. Panel A is a negative controlrepresenting untransfected CHO cells.

FIG. 22 depicts flow cytometric profiles of binding of control Ig,B7-1Ig, B7-2Ig, B7-2VIg, and anti-CD28 to CHO cells expressing CD28.

FIG. 23 represents a histogram showing proliferation of CD28+ T cellsstimulated with 1 ng/ml PMA alone or with either of the followingcostimulatory signals: CHO/B7-1 cells, CHO/B7-2 cells, control Ig (30μg/ml), or B7-1Ig, B7-2Ig, or B7-2VIg (30 μg or 100 μg/ml each).

FIG. 24 represents a histogram showing proliferation of, and IL-2production by CD28+ T cells incubated with anti-CD3 attached to platesand B7-1Ig (10, 3 or 1 μg/ml) or B7-2Ig (19, 3 or 1 μg/ml) or B7-2VIg(3.0-0.01 μg/ml).

FIG. 25 represents the amount of IL-2 produced by CD28+ T cells after 1,2, or 3 days of incubation of the cells with anti-CD3 alone or togetherwith either CHO/B7-2 cells or B7-2VIg fusion protein.

FIG. 26 represents the amount of IL-2 secreted by CD28+ T cells after 1,2, or 5 days of incubation of the cells with anti-CD3 alone or witheither anti-CD28, B7-1Ig, B7-2Ig, or B7-2VIg.

FIG. 27 is a graphical representation of the growth of CD28+ T cellsincubated with anti-CD3 alone, or with B7-1Ig, B7-2Ig, B7-2VIg, ortogether with either anti-CD28.

DETAILED DESCRIPTION OF THE INVENTION

In addition to the previously characterized B lymphocyte activationantigen B7 (referred to herein as B7-1), human B lymphocytes expressother novel molecules which costimulate T cell activation. Thesecostimulatory molecules include antigens on the surface of Blymphocytes, professional antigen presenting cells (e.g., monocytes,dendritic cells, Langerhan cells) and other cells (e.g., keratinocytes,endothelial cells, astrocytes, fibroblasts, oligodendrocytes) whichpresent antigen to immune cells, and which bind either CTLA4, CD28, bothCTLA4 and CD28 or other known or as yet undefined receptors on immunecells. Costimulatory molecules within the scope of the invention arereferred to herein as CTLA4/CD28 ligands (counter-receptors) or Blymphocyte antigens. Novel B lymphocyte antigens which providecotimulation to activated T cells to thereby induce T cell proliferationand/or cytokine secretion include the B7-2 (human and murine) and theB7-3 antigens described and characterized herein.

The B lymphocyte antigen B7-2 is expressed by human B cells at about 24hours following stimulation with either anti-immunoglobulin or anti-MHCclass II monoclonal antibody. The B7-2 antigen induces detectable IL-2secretion and T cell proliferation. At about 48 to 72 hours postactivation, human B cells express both B7-1 and a third CTLA4counter-receptor, B7-3, identified by a monoclonal antibody BB-1, whichalso binds B7-1 (Yokochi, T., et al. (1982) J. Immunol. 128, 823-827).The B7-3 antigen is also expressed on B7-1 negative activated B cellsand can costimulate T cell proliferation without detectable IL-2production, indicating that the B7-1 and B7-3 molecules are distinct.B7-3 is expressed on a wide variety of cells including activated Bcells, activated monocytes, dendritic cells, Langerhan cells andkeratinocytes. At 72 hours post B cell activation, the expression ofB7-1 and B7-3 begins to decline. The presence of these costimulatorymolecules on the surface of activated B lymphocytes indicates that Tcell costimulation is regulated, in part, by the temporal expression ofthese molecules following B cell activation.

Accordingly, one aspect of this invention pertains to isolated nucleicacids comprising a nucleotide sequence encoding a novel costimulatorymolecule, such as the B lymphocyte antigen, B7-2, fragments of suchnucleic acids, or equivalents thereof. The term "nucleic acid" as usedherein is intended to include such fragments or equivalents. The term"equivalent" is intended to include nucleotide sequences encodingfunctionally equivalent B lymphocyte antigens or functionally equivalentpeptides having an activity of a novel B lymphocyte antigen, i.e., theability to bind to the natural ligand(s) of the B lymphocyte antigen onimmune cells, such as CTLA4 and/or CD28 on T cells, and inhibit (e.g.,block) or stimulate (e.g., enhance) immune cell costimulation. Suchnucleic acids are considered equivalents of the human B7-2 nucleotidesequence provided in FIG. 8 (SEQ ID NO:1) and the murine B7-2 nucleotidesequence provided in FIG. 14 (SEQ ID NO:22) and are within the scope ofthis invention.

In one embodiment, the nucleic acid is a cDNA encoding a peptide havingan activity of the B7-2 B lymphocyte antigen. Preferably, the nucleicacid is a cDNA molecule consisting of at least a portion of a nucleotidesequence encoding human B7-2, as shown in FIG. 8 (SEQ ID NO:1) or atleast a portion of a nucleotide sequence encoding murine B7-2, as shownin FIG. 14 (SEQ ID NO:22). A preferred portion of the cDNA molecule ofFIG. 8 (SEQ ID NO:1) or FIG. 14 (SEQ ID NO:22) includes the codingregion of the molecule.

In another embodiment, the nucleic acid of the invention encodes apeptide having an activity of B7-2 and comprising an amino acid sequenceshown in FIG. 8 (SEQ ID NO:2) or FIG. 14 (SEQ ID NO:23). Preferrednucleic acids encode a peptide having B7-2 activity and at least about50% homology, more preferably at least about 60% homology and mostpreferably at least about 70% homology with an amino acid sequence shownin FIG. 8 (SEQ ID NO:2). Nucleic acids which encode peptides having B7-2activity and at least about 90%, more preferably at least about 95%, andmost preferably at least about 98-99% homologous with a sequence setforth in FIG. 8 (SEQ ID NO:2) are also within the scope of theinvention. Homology refers to sequence similarity between two peptideshaving the activity of a novel B lymphocyte antigen, such as B7-2, orbetween two nucleic acid molecules. Homology can be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequences is occupied bythe same nucleotide base or amino acid, then the molecules arehomologous at that position. A degree (or percentage) of homologybetween sequences is a function of the number of matching or homologouspositions shared by the sequences.

Another aspect of the invention provides a nucleic acid which hybridizesunder high or low stringency conditions to a nucleic acid which encodesa peptide having all or a portion of an amino acid sequence shown inFIG. 8 (SEQ ID NO:2) or a peptide having all or a portion of an aminoacid sequence shown in FIG. 14 (SEQ ID NO:23). Appropriate stringencyconditions which promote DNA hybridization, for example, 6.0×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. are known to those skilled in the art or can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. For example, the salt concentration in the washstep can be selected from a low stringency of about 2.0×SSC at 50° C. toa high stringency of about 0.2×SSC at 50° C. In addition, thetemperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C. to high stringencyconditions, at about 65° C.

Isolated nucleic acids encoding a peptide having an activity of a novelB lymphocyte antigen, as described herein, and having a sequence whichdiffers from nucleotide sequence shown in FIG. 8 (SEQ ID NO:1) or FIG.14 (SEQ ID NO:22) due to degeneracy in the genetic code are also withinthe scope of the invention. Such nucleic acids encode functionallyequivalent peptides (e.g., a peptide having B7-2 activity) but differ insequence from the sequence of FIG. 8 or FIG. 14 due to degeneracy in thegenetic code. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC are synonyms for histidine) may occurdue to degeneracy in the genetic code. As one example, DNA sequencepolymorphisms within the nucleotide sequence of a B7-2 (especially thosewithin the third base of a codon) may result in "silent" mutations inthe DNA which do not affect the amino acid encoded. However, it isexpected that DNA sequence polymorphisms that do lead to changes in theamino acid sequences of the B7-2 antigen will exist within a population.It will be appreciated by one skilled in the art that these variationsin one or more nucleotides (up to about 3-4% of the nucleotides) of thenucleic acids encoding peptides having the activity of a novel Blymphocyte antigen may exist among individuals within a population dueto natural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of theinvention. Furthermore, there may be one or more isoforms or related,cross-reacting family members of the novel B lymphocyte antigensdescribed herein. Such isoforms or family members are defined asproteins related in function and amino acid sequence to a B lymphocyteantigen (e.g., the B7-2 antigen), but encoded by genes at differentloci.

A "fragment" of a nucleic acid encoding a novel B lymphocyte antigen isdefined as a nucleotide sequence having fewer nucleotides than thenucleotide sequence encoding the entire amino acid sequence of the Blymphocyte antigen and which encodes a peptide having an activity of theB lymphocyte antigen (i.e., the ability to bind to the natural ligand(s)of the B lymphocyte antigen on immune cells, such as CTLA4 and/or CD28on T cells and either stimulate or inhibit immune cell costimulation).Thus, a peptide having B7-2 activity binds CTLA4 and/or CD28 andstimulates or inhibits a T cell mediated immune response, as evidencedby, for example, cytokine production and/or T cell proliferation by Tcells that have received a primary activation signal. In one embodiment,the nucleic acid fragment encodes a peptide of the B7-2 antigen whichretains the ability of the antigen to bind CTLA4 and/or CD28 and delivera costimulatory signal to T lymphocytes. In another embodiment, thenucleic acid fragment encodes a peptide including an extracellularportion of the human B7-2 antigen (e.g., approximately amino acidresidues 24-245 of the sequence provided in FIG. 8 (SEQ ID NO:2)) whichcan be used to bind CTLA4 and/or CD28 and, in monovalent form, inhibitcostimulation, or in multivalent form, induce or enhance costimulation.

Preferred nucleic acid fragments encode peptides of at least 20 aminoacid residues in length, preferably at least 40 amino acid residues andlength, and more preferably at least 60 amino acid residues in length.Nucleic acid fragments which encode peptides of at least 80 amino acidresidues in length, at least 100 amino acid residues in length, and atleast 200 or more amino acids in length are also within the scope of theinvention. Particularly preferred nucleic acid fragments encode apeptide having the activity of human B7-2 and an amino acid sequencerepresented by a formula:

    X.sub.n --Y--Z.sub.m

In the fomula, Y comprises amino acid residues 24-245 of the sequenceshown in FIG. 8 (SEQ ID NO:2). X_(n) and Z_(m) are additional amino acidresidue(s) linked to Y by an amide bond. X_(n) and Z_(m) are selectedfrom amino acid residues contiguous to Y in the amino acid sequenceshown in FIG. 8 (SEQ ID NO:2). In the formula, X_(n) is amino acidresidue(s) selected from amino acids contiguous to the amino terminus ofY in the sequence shown in FIG. 8 (SEQ ID NO:2), i.e., from amino acidresidue 23 to 1. Z_(m) is amino acid residue(s) selected from aminoacids contiguous to the carboxy terminus of Y in the sequence shown inFIG. 8 (SEQ ID NO:2), i.e., from amino acid residue 246 to 329. Inaddition, in the formula, n is a number from 0 to 23 (n=0-23) and m is anumber from 0 to 84 (m=0-84). A particularly preferred peptide has anamino acid sequence represented by the formula X_(n) --Y--Z_(m) asabove, where n=0 and m=0.

Nucleic acid fragments within the scope of the invention include thosecapable of hybridizing with nucleic acid from other animal species foruse in screening protocols to detect novel proteins that arecross-reactive with the B lymphocyte antigens described herein. Theseand other fragments are described in detail herein. Generally, thenucleic acid encoding a fragment of a B lymphocyte antigen will beselected from the bases coding for the mature protein, however, in someinstances it may be desirable to select all or part of a fragment orfragments from the leader sequence or non-coding portion of a nucleotidesequence. Nucleic acids within the scope of the invention may alsocontain linker sequences, modified restriction endonuclease sites andother sequences useful for molecular cloning, expression or purificationof recombinant protein or fragments thereof. These and othermodifications of nucleic acid sequences are described in further detailherein.

A nucleic acid encoding a peptide having an activity of a novel Blymphocyte antigen, such as the B7-2 antigen, may be obtained from mRNApresent in activated B lymphocytes. It should also be possible to obtainnucleic acid sequences encoding B lymphocyte antigens from B cellgenomic DNA. For example, the gene encoding the B7-2 antigen can becloned from either a cDNA or a genomic library in accordance withprotocols herein described. A cDNA encoding the B7-2 antigen can beobtained by isolating total mRNA from an appropriate cell line. Doublestranded cDNAs can then prepared from the total mRNA. Subsequently, thecDNAs can be inserted into a suitable plasmid or viral (e.g.,bacteriophage) vector using any one of a number of known techniques.Genes encoding novel B lymphocyte antigens can also be cloned usingestablished polymerase chain reaction techniques in accordance with thenucleotide sequence information provided by the invention. The nucleicacids of the invention can be DNA or RNA. A preferred nucleic acid is acDNA encoding the human B7-2 antigen having the sequence depicted inFIG. 8 (SEQ ID NO:1). Another preferred nucleic acid is a cDNA encodingthe murine B7-2 antigen having the sequence shown on FIG. 14 (SEQ IDNO:22).

This invention further pertains to expression vectors containing anucleic acid encoding at least one peptide having the activity of anovel B lymphocyte antigen, as described herein, operably linked to atleast one regulatory sequence. "Operably linked" is intended to meanthat the nucleotide acid sequence is linked to a regulatory sequence ina manner which allows expression of the nucleotide sequence (e.g., incis or trans). Regulatory sequences are art-recognized and are selectedto direct expression of the desired protein in an appropriate host cell.Accordingly, the term regulatory sequence includes promoters, enhancersand other expression control elements. Such regulatory sequences areknown to those skilled in the art or one described in Goeddel, GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transfected and/or the type of protein desired to beexpressed. In one embodiment, the expression vector includes a nucleicacid encoding at least a portion of the B7-2 protein, such as anextracellular domain portion. In another embodiment, the expressionvector includes a DNA encoding a peptide having an activity of the B7-2antigen and a DNA encoding a peptide having an activity of another Blymphocyte antigen, such as B7-1. cDNAs encoding the human B7-1 andmouse B7-1 antigens are shown in SEQ ID NO:28 and SEQ ID NO:30,respectively. The deduced amino acid sequences of these antigens arealso shown in SEQ ID NO:29 and SEQ ID NO:31, respectively. Suchexpression vectors can be used to transfect cells to thereby produceproteins or peptides, including fusion proteins or peptides encoded bynucleic acid sequences as described herein. These and other embodimentsare described in further detail herein.

The invention also features methods of producing peptides having anactivity of a novel B lymphocyte antigen. For example, a host celltransfected with a nucleic acid vector directing expression of anucleotide sequence encoding a peptide having an activity of the B7-2protein can be cultured in a medium under appropriate conditions toallow expression of the peptide to occur. In addition, one or moreexpression vectors containing DNA encoding a peptide having an activityof B7-2 and DNA encoding another peptide, such as a peptide having anactivity of a second B lymphocyte antigen (e.g., B7-1, B7-3) can be usedto transfect a host cell to coexpress these peptides or produce fusionproteins or peptides. In one embodiment, a recombinant expression vectorcontaining DNA encoding a B7-2 fusion protein is produced. A B7-2 fusionprotein can be produced by recombinant expression of a nucleotidesequence encoding a first peptide having B7-2 activity and a nucleotidesequence encoding second peptide corresponding to a moiety that altersthe solubility, affinity, stability or valency of the first peptide, forexample, an immunoglobulin constant region. Preferably, the firstpeptide consists of a portion of the extracellular domain of the humanB7-2 antigen (e.g., approximately amino acid residues 24-245 of thesequence shown in FIG. 8 (SEQ ID NO:2)). The second peptide can includean immunoglobulin constant region, for example, a human Cγ1 domain orCγ4 domain (e.g., the hinge, CH2 and CH3 regions of human IgCγ1, orhuman IgCγ4, see e.g., Capon et al. U.S. Pat. No. 5,116,964,incorporated herein by reference). A resulting B7-2Ig fusion protein mayhave altered B7-2 solubility, binding affinity, stability and/or valency(i.e., the number of binding sites available per molecule) and mayincrease the efficiency of protein purification. Fusion proteins andpeptides produced by recombinant technique may be secreted and isolatedfrom a mixture of cells and medium containing the protein or peptide.Alternatively, the protein or peptide may be retained cytoplasmicallyand the cells harvested, lysed and the protein isolated. A cell culturetypically includes host cells, media and other byproducts. Suitablemediums for cell culture are well known in the art. Protein and peptidescan be isolated from cell culture medium, host cells, or both usingtechniques known in the art for purifying proteins and peptides.Techniques for transfecting host cells and purifying proteins andpeptides are described in further detail herein.

Particularly preferred human B7-2Ig fusion proteins include theextracellular domain portion or variable region-like domain of humanB7-2 coupled to an immunoglobulin constant region. The immunoglobulinconstant region may contain genetic modifications which reduce oreliminate effector activity inherent in the immunoglobulin structure.For example, DNA encoding the extracellular portion of human B7-2(hB7-2), as well as DNA encoding the variable region-like domain ofhuman B7-2 (hB7.2V) or the constant region-like domain of human B7-2(hB7.2C) can be joined to DNA encoding the hinge, CH2 and CH3 regions ofhuman IgCγ1 and/or IgCγ4 modified by site directed mutagenesis. Thepreparation and characterization of these fusion proteins is describedin detail in Example 7.

In a specific embodiment, the protein of the invention is a variableregion form of the B cell activation antigen B7-2. The language "avariable region form of the B cell activation antigen B7-2" is intendedto include forms of B7-2 which contain the immunoglobulin-like variabledomain of B7-2, but do not comprise the immunoglobulin-like constantdomain of B7-2. In a preferred embodiment, the variable region form ofB7-2 comprises an amino acid sequence starting at about amino acidpostion 18 to 30 and ending about amino acid position 128 to 140 ofhuman B7-2 protein (SEQ ID NO:2). In a most preferred embodiment, thevariable form of B7-2 comprises about amino acids 24 to 133 of humanB7-2 protein (SEQ ID NO:2). The variable region form of B7-2 can furtherbe operatively linked directly to a transmembrane domain, such as thetransmembrane domain of B7-2, to form a variable region form of B7-2that can be expressed on a cell surface. "Operatively" is intended tomean in such a way that the molecule formed by operatively linking twoor more domains or peptides is functional. The transmembrane domain ofhuman B7-2 comprises about amino residues 246 to 268 of human B7-2protein. Thus, in one embodiment, the variable region form of B7-2 isoperatively linked to a peptide having a first amino acid locatedbetween about amino acid residue 238 and about amino acid residue 252,and a last amino acid residue located between about amino acid residue260 and about amino acid residue 274 of human B7-2 protein of sequenceSEQ ID NO:2. The incorporation of a transmembrane domain in a protein ofthe invention, allows the protein to be expressed on a cell surface whena nucleic acid encoding the protein is expressed in the cell.

In another embodiment, the variable region form of B7-2 s operativelylinked to a cytoplasmic domain, such as a cytoplasmic domain of B7-2.The cytoplasmic domain of human B7-2 comprises about amino acid residues269 to 329 of human B7-2 protein of SEQ ID NO:2. Accordingly, in oneembodiment, the variable region form of B7-2 is operatively linked to asecond B7-2 peptide, having a first amino acid residue located betweenabout amino acids 260 and 275 of human B7-2 and a terminal amino acidresidue located between about amino acid 323 to about amino acid 335 ofhuman B7-2 of SEQ ID NO:2. In another embodiment, a variable region formof B7-2 is operatively linked to a second B7-2 peptide of about aminoacid residues 269 to 329 of human B7-2.

In a further embodiment, a variable region form of B7-2 operativelylinked to a peptide corresponding to about the transmembrane domain ofB7-2 is further operatively linked to a peptide correspondingsignificantly to the cytoplasmic domain of B7-2. Thus, proteins withinthe scope of the invention include those comprising an amino acidsequence from about position 24 to about position 133 of SEQ ID NO:2,operatively linked to an amino acid sequence from about position 246 toabout position 268 of SEQ ID NO:2 (V-region and transmembrane domains).Other proteins within the scope of the invention include thosecomprising an amino acid sequence from about position 24 to aboutposition 133 of SEQ ID NO:2, operatively linked to an amino acidsequence from about position 246 to about position 329 of SEQ ID NO:2(V-region, transmembrane and cytoplasmic domains). Yet other proteinswithin the scope of the invention include the leader sequence of B7-2(e.g. positions 1-23) at the N-terminus. Proteins including otherportions of B7-2 protein operatively linked to each other, but notincluding the immunoglobulin-like constant domain of B7-2, are alsowithin the scope of the invention. In other embodiments, B7-2 proteinsthat contain an immunoglobulin-like constant domain of B7-2 in theabsence of the variable region are contemplated.

A variable region form of B7-2, can also be linked to at least oneheterologous polypeptide. The term "heterologous polypeptide" isintended to include any polypeptide, such as a polypeptide that directsthe protein of the invention to a specific cellular compartment. In oneembodiment, the heterologous polypeptide is a signal peptide that allowsfor the protein to be secreted from the cell. Another heterologouspolypeptide within the scope of the invention is a signal peptide thatallows for the protein to be expressed on the surface of the cell. Inyet another embodiment, the heterologous polypeptide is a constantregion of an immunoglobulin molecule. In an even more preferredembodiment, the heterologous polypeptide comprises the hinge, CH2, andCH3 domains of IgG1, as described herein.

The variable region form of B7-2 can further be attached to a linkerpolypeptide. A "linker polypeptide" as defined herein includes anypolypeptide that bridges two peptides in the protein of the invention.Alternatively, the linker peptide is attached to either or both ends ofthe protein. Thus, a linker peptide attached to one or both ends of theprotein can for example facilitate binding of the protein of theinvention to a solid support. The linker peptide can also be a fragmentof a bacterial or viral protein.

The fusion proteins described above can be, for example, human ormurine. The nucleic acid molecules encoding the above described fusionproteins, as well as expression vectors and host cells expressing thefusion proteins are also within the scope of the invention.

Transfected cells which express peptides having an activity of one ormore B lymphocyte antigens (e.g., B7-2, B7-3) on the surface of the cellare also within the scope of this invention. In one embodiment, a hostcell such as a COS cell is transfected with an expression vectordirecting the expression of a peptide having B7-2 activity on thesurface of the cell. Such a transfected host cell can be used in methodsof identifying molecules which inhibit binding of B7-2 to itscounter-receptor on T cells or which interfere with intracellularsignaling of costimulation to T cells in response to B7-2 interaction.In another embodiment, a tumor cell such as a sarcoma, a melanoma, aleukemia, a lymphoma, a carcinoma or a neuroblastoma is transfected withan expression vector directing the expression of at least one typicallyat least 20 amino acid residues in length, preferably at least 40 aminoacid residues in length, and most preferably 60 amino acid residues inlength. Peptides having B7-2 activity and including at least 80 aminoacid residues in length, at least 100 amino acid residues in length, orat least 200 or more amino acid residues in length are also within thescope of the invention. A preferred peptide includes an extracellulardomain portion of the human B7-2 antigen (e.g., about amino acidresidues 24-245 of the sequence shown in FIG. 8 (SEQ ID NO:2). Otherpreferred peptides have an amino acid sequence represented by a formula:

    X.sub.n --Y--Z.sub.m

where Y is amino acid residues selected from the group consisting of:amino acid residues 55-68 of the sequence shown in FIG. 8 (SEQ ID NO:2);amino acid residues 81-89 of the sequence shown in FIG. 8 (SEQ ID NO:2);amino acid residues 128-142 of the sequence shown in FIG. 8 (SEQ IDNO:2); amino acid residues 160-169 of the sequence shown in FIG. 8 (SEQID NO:2); amino acid residues 188-200 of the sequence shown in FIG. 8(SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown inFIG. 8 (SEQ ID NO:2). In the formula, X_(n) and Z_(m) are additionalamino acid residues linked to Y by an amide bond. X_(n) and Z_(m) areamino acid residues selected from amino acids contiguous to Y in theamino acid sequence shown in FIG. 8 (SEQ ID NO:2). X_(n) is amino acidresidues selected from amino acids contiguous to the amino terminus of Yin the sequence shown in FIG. 8 (SEQ ID NO:2). Z_(m) is amino acidresidues selected from amino acids contiguous to the carboxy terminus ofY in the sequence shown in FIG. 8 (SEQ ID NO:2). According to theformula, n is a number from 0 to 30 (n=0-30) and m is a number from 0 to30 (m=0-30). A particularly preferred peptide has an amino acid sequencerepresented by the formula X_(n) --Y--Z_(m), where n=0 and m=0.

Another embodiment of the invention provides a substantially purepreparation of a peptide having an activity of a novel B lymphocyteantigen such as B7-2 or B7-3. Such a preparation is substantially freeof proteins and peptides with which the peptide naturally occurs in acell or with which it naturally occurs when secreted by a cell.

The term "isolated" as used throughout this application refers to anucleic acid, protein or peptide having an activity of a novel Blymphocyte antigen, such as B7-2, substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.An isolated nucleic acid is also free of sequences which naturally flankthe nucleic acid (i.e., sequences located at the 5' and 3' ends of thenucleic acid) in the organism from which the nucleic acid is derived.peptide having the activity of a novel B lymphocyte antigen on thesurface of the tumor cell. In some instances, it may be beneficial totransfect a tumor cell to coexpress major histocompatibility complex(MHC) proteins, for example MHC class II α and β chain proteins or anMHC class I α chain protein, and, if necessary, a β2 microglobulinprotein. Such transfected tumor cells can be used to induce tumorimmunity in a subject. These and other embodiments are described infurther detail herein.

The nucleic acid sequences of the invention can also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al U.S.Pat. No. 4,598,049; Caruthers t al U.S. Pat. No. 4,458,066; and ItakuraU.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated by referenceherein).

Another aspect of the invention pertains to isolated peptides having anactivity of a novel B lymphocyte antigen (e.g., B7-2, B7-3). A peptidehaving an activity of a B lymphocyte antigen may differ in amino acidsequence from the B lymphocyte antigen, such as the human B7-2 sequencedepicted in FIG. 8 (SEQ ID NO:2), or murine B7-2 sequence depicted inFIG. 14 (SEQ ID NO:22), but such differences result in a peptide whichfunctions in the same or similar manner as the B lymphocyte antigen orwhich has the same or similar characteristics of the B lymphocyteantigen. For example, a peptide having an activity of the B7-2 proteinis defined herein as a peptide having the ability to bind to the naturalligand(s) of the B7-2 protein on immune cells, such as CLTA4 and/or CD28on T cells and either stimulate or inhibit immune cell costimulation.Thus, a peptide having B7-2 activity binds CTLA4 and/or CD28 andstimulates or inhibits a T cell mediated immune response (as evidencedby, for example, cytokine production and/or proliferation by T cellsthat have received a primary activation signal). One embodiment providesa peptide having B7-2 binding activity, but lacking the ability todeliver a costimulatory signal to T cells. Such a peptide can be used toinhibit or block T cell proliferation and/or cytokine secretion in asubject. Alternatively, a peptide having both B7-2 binding activity andthe ability to deliver a costimulatory signal to T cells is used tostimulate or enhance T cell proliferation and/or cytokine secretion in asubject. Various modifications of the B7-2 protein to produce these andother functionally equivalent peptides are described in detail herein.The term "peptide" as used herein, refers to peptides, proteins andpolypeptides.

A peptide can be produced by modification of the amino acid sequence ofthe human B7-2 protein shown in FIG. 8 (SEQ ID NO:2) or the murine B7-2protein shown in FIG. 14 (SEQ ID NO:23), such as a substitution,addition or deletion of an amino acid residue which is not directlyinvolved in the function of B7-2 (i.e., the ability of B7-2 to bindCTLA4 and/or CD28 and/or stimulate or inhibit T cell costimulation).Peptides of the invention are

The various peptides, polypeptides, proteins, and fusion proteins of theinvention can be prepared as soluble forms. Alternatively, the proteinsof the invention can be expressed on the surface of a cell, such as aCHO cell and can be prepared according to methods well known in the art.The proteins of the invention can also be coupled to a solid phasesupport, such as a bead or a plate. In a specific embodiment, a B7-2Igfusion protein is attached to a solid phase support, such as a bead, forexample a biodegradable bead. In a preferred embodiment, a variableregion form of B7-2 is attached to a solid support. In a most preferredembodiment, a B7-2VIg fusion protein comprising an amino acid sequenceof about position 24 to 133 of human B7-2Ig (SEQ ID NO:2) linked to theconstant domain of an IgG molecule is attached to a solid phase support.

These molecules can then be attached to a solid phase surface viaseveral possible methods. For example, the proteins of the invention,such as a variable region form of B7-2, can be crosslinked to the beadsvia covalent modification using tosyl linkage. In this method, theproteins of the invention are typically in 0.05M borate buffer, pH 9.5and added to tosyl-activated magnetic immunobeads (Dynal Inc., GreatNeck, N.Y.) according to manufacturer's instructions. After a 24 hrincubation at 22° C., the beads are collected and washed extensively. Itis not mandatory that immunomagnetic beads be used, as other methods arealso satisfactory. For example, proteins of the invention may also beimmobilized on polystyrene beads or culture vessel surfaces.

It is also possible to attach the proteins, such as B7-2VIg to a solidphase surface through an avidin- or streptavidin-biotin complex. In thisparticular embodiment, the soluble protein is first crosslinked tobiotin and then reacted with the solid phase surface to which avidin orstreptavidin molecules are bound. It is also possible to crosslink theprotein with avidin or streptavidin and to react these with a solidphase surface that is covered with biotin molecules.

These and other aspects of this invention are described in detail in thefollowing subsections.

I. Isolation of Nucleic Acid From Cell Lines

Suitable cells for use in isolating nucleic acids encoding peptideshaving an activity of a novel B lymphocyte antigen include cells capableof producing mRNA coding for B lymphocyte antigens (e.g., B7-1, B7-2,B7-3) and appropriately translating the mRNA into the correspondingprotein. One source of mRNA is normal human splenic B cells, eitherresting or activated by treatment with an anti-immunoglobulin antibodyor an anti-MHC class II antibody, or from subsets of neoplastic B cells.Expression of the human B7-2 antigen is detectable in resting B cellsand in activated B cells, with mRNA levels increasing 4-fold fromresting levels following stimulation. Total cellular RNA can be obtainedusing standard techniques from resting or activated B cells during theseintervals and utilized in the construction of a cDNA library.

In addition, various subsets of neoplastic B cells may express B7-2 andB7-3 and can alternatively serve as a source of the mRNA forconstruction of a cDNA library. For example, tumor cells isolated frompatients with non-Hodgkins lymphoma express B7-1 mRNA. B cells fromnodular, poorly differentiated lymphoma (NPDL), diffuse large celllymphoma (LCL) and Burkitt's lymphoma cell lines are also suitablesources of human B7-1 mRNA and, potentially B7-2 and B7-3 mRNA. Myelomasgenerally express B7-2, but not B7-1 mRNA, and, thus can provide asource of B7-2 mRNA. The Burkitt's lymphoma cell line Raji is one sourceof B lymphocyte antigen mRNA. Preferably, B7-2 mRNA is obtained from apopulation of both resting and activated normal human B cells. ActivatedB cells can be obtained by stimulation over a broad spectrum of time(e.g., from minutes to days) with, for example, an anti-immunoglobulinantibody or an anti-MCH class II antibody.

II. Isolation of mRNA and Construction of cDNA Library

Total cellular mRNA can be isolated by a variety of techniques, e.g., byusing the guanidinium-thiocyanate extraction procedure of Chirgwin etal., Biochemistry 18, 5294-5299 (1979). According to this method, Poly(A+) mRNA is prepared and purified for use in a cDNA libraryconstruction using oligo (dT) cellulose selection. cDNA is thensynthesized from the poly (A+) RNA using oligo (dT) priming and reversetranscriptase. Moloney MLV reverse transcriptase (available fromGibco/BRL, Bethesda, Md.) or AMV reverse transcriptase (available fromSeikagaku America, Inc., St. Petersburg, Fla.) are preferably employed.

Following reverse transcription, the mRNA/DNA hybrid molecule isconverted to double stranded DNA using conventional techniques andincorporated into a suitable vector. The experiments herein employed E.coli DNA polymerase I and ribonuclease H in the conversion to doublestranded cDNA.

Cloning of the cDNAs can be accomplished using any of the conventionaltechniques for joining double stranded DNA with an appropriate vector.The use of synthetic adaptors is particularly preferred, since italleviates the possibility of cleavage of the cDNA with restrictionenzyme prior to cloning. Using this method, non-self complementary,kinased adaptors are added to the DNA prior to ligation with the vector.Virtually any adaptor can be employed. As set forth in more detail inthe examples below, non-self complementary BstXI adaptors are preferablyadded to the cDNA for cloning, for ligation into a pCDM8 vector preparedfor cloning by digestion with BstXI.

Eucaryotic cDNA can be expressed when placed in the sense orientation ina vector that supplies an appropriate eucaryotic promoter and origin ofreplication and other elements including enhancers, splice acceptorsand/or donor sequences and polyadenylation signals. The cDNAs of thepresent invention are placed in suitable vectors containing a eucaryoticpromoter, an origin of replication functional in E. coli, an SV40 originof replication which allows growth in COS cells, and a cDNA insertionsite. Suitable vectors include πH3 (Seed and Aruffo, Proc. Natl. Acad.Sci., 84:3365-3369 (1987)), πH3m (Aruffo and Seed, Proc. Natl. AcadSci., 84:8573-8577 (1987)), pCDM7 and pCDM8 (Seed, Nature, 329:840-841(1987), with the pCDM8 vector being particularly preferred (availablecommercially from Invitrogen, San Diego, Calif.).

III. Transfection of Host Cells and Screening for Novel B LymphocyteActivation Antigens

The thus prepared cDNA library is then used to clone the gene ofinterest by expression cloning techniques. A basic expression cloningtechnique has been described by Seed and Aruffo, Proc. Natl. Acad. Sci.USA, 84:3365-3369 (1987) and Aruffo and Seed, Proc. Natl. Acad. Sci.USA, 84:8573-8577 (1987), although modifications to this technique maybe necessary.

According to one embodiment, plasmid DNA is introduced into a simian COScell line (Gluzman, Cell 23:175 (1981)) by known methods of transfection(e.g., DEAE-Dextran) and allowed to replicate and express the cDNAinserts. The transfectants expressing B7-1 antigen are depleted with ananti-B7-1 monoclonal antibody (e.g., 133 and B1.1) and anti-murine IgGand IgM coated immunomagnetic beads. Transfectants expressing human B7-2antigen can be positively selected by reacting the transfectants withthe fusion proteins CTLA4Ig and CD28Ig, followed by panning withanti-human Ig antibody coated plates. Although human CTLA4Ig and CD28Igfusion proteins were used in the examples described herein, given thecross-species reactivity between B7-1 and, for example murine B7-1, itcan be expected that other fusion proteins reactive with anothercross-reactive species could be used. After panning, episomal DNA isrecovered from the panned cells and transformed into a competentbacterial host, preferably E. coli. Plasmid DNA is subsequentlyreintroduced into COS cells and the cycle of expression and panningrepeated at least two times. After the final cycle, plasmid DNA isprepared from individual colonies, transfected into COS cells andanalyzed for expression of novel B lymphocyte antigens by indirectimmunofluorescence with, for example, CTLA4Ig and CD28Ig.

IV. Sequencing of Novel B Lymphocyte Antigens

Plasmids are prepared from those clones which are strongly reactive withthe CTLA4Ig and/or CD28Ig. These plasmids are then sequenced. Any of theconventional sequencing techniques suitable for sequencing tracts of DNAabout 1.0 kb or larger can be employed.

As described in Example 4, a human B7-2 clone (clone29) was obtainedcontaining an insert of 1,120 base pairs with a single long open readingframe of 987 nucleotides and approximately 27 nucleotides of 3'noncoding sequences (FIG. 8, SEQ ID NO:1). The predicted amino acidsequence encoded by the open reading frame of the protein is shown belowthe nucleotide sequence in FIG. 8. The encoded human B7-2 protein, ispredicted to be 329 amino acid residues in length (SEQ ID NO:2). Thisprotein sequence exhibits many features common to other type I Igsuperfamily membrane proteins. Protein translation is predicted to beginat the methionine codon (ATG, nucleotides 107 to 109) based on the DNAhomology in this region with the consensus eucaryotic translationinitiation site (see Kozak, M. (1987) Nucl. Acids Res. 15:8125-8148).The amino terminus of the B7-2 protein (amino acids 1 to 23) has thecharacteristics of a secretory signal peptide with a predicted cleavagebetween the alanines at positions 23 and 24 (von Heijne (1987) Nucl.Acids Res. 14:4683). Processing at this site would result in a B7-2membrane bound protein of 306 amino acids having an unmodified molecularweight of approximately 34 kDa. This protein would consist of anapproximate extracellular Ig superfamily V and C like domains of fromabout amino acid residue 24 to 245, a hydrophobic transmembrane domainof from about amino acid residue 246 to 268, and a long cytoplasmicdomain of from about amino acid residue 269 to 329. The homologies tothe Ig superfamily are due to the two contiguous Ig-like domains in theextracellular region bound by the cysteines at positions 40 to 110 and157 to 218. The extracellular domain also contains eight potentialN-linked glycosylation sites and, like B7-1, is probably glycosylated.Glycosylation of the human B7-2 protein may increase the molecularweight to about 50-70 kDa. The cytoplasmic domain of human B7-2, whilesomewhat longer than B7-1, contains a common region of multiplecysteines followed by positively charged amino acids which presumablyfunction as signaling or regulatory domains within an antigen-presentingcell (APC). Comparison of both the nucleotide and amino acid sequencesof the human B7-2 with the GenBank and EMBL databases yieldedsignificant homology (about 26% amino acid sequence identity) with humanB7-1. Since human B7-1, human B7-2 and murine B7-1 all bind to humanCTLA4 and CD28, the homologous amino acids probably represent thosenecessary to comprise a CTLA4 or CD28 binding sequence. E. colitransfected with a vector containing a cDNA insert encoding human B7-2(clone 29) was deposited with the American Type Culture Collection(ATCC) on Jul. 26, 1993 as Accession No. 69357.

V. Cloning Novel B Lymphocyte Antigens from Other Mammalian Species

The present invention is not limited to human nucleic acid molecules andcontemplates that novel B lymphocyte antigen homologues from othermammalian species that express B lymphocyte antigens can be cloned andsequenced using the techniques described herein. B lymphocyte antigensisolated for one species (e.g., humans) which exhibit cross-speciesreactivity may be used to modify T cell mediated immune responses in adifferent species (e.g., mice). Isolation of cDNA clones from otherspecies can also be accomplished using human cDNA inserts, such as humanB7-2 cDNA, as hybridization probes.

As described in Example 6, a murine B7-2 clone (mB7-2, clone 4) wasobtained containing an insert of 1,163 base pairs with a single longopen reading frame of 927 nucleotides and approximately 126 nucleotidesof 3' noncoding sequences (FIG. 14, SEQ ID NO:22). The predicted aminoacid sequence encoded by the open reading frame of the protein is shownbelow the nucleotide sequence in FIG. 14. The encoded murine B7-2protein, is predicted to be 309 amino acid residues in length (SEQ IDNO:23). This protein sequence exhibits many features common to othertype I Ig superfamily membrane proteins. Protein translation ispredicted to begin at the methionine codon (ATG, nucleotides 111 to 113)based on the DNA homology in this region with the consensus eucaryotictranslation initiation site (see Kozak, M. (1987) Nucl. Acids Res.15:8125-8148). The amino terminus of the murine B7-2 protein (aminoacids 1 to 23) has the characteristics of a secretory signal peptidewith a predicted cleavage between the alanine at position 23 and thevaline at position 24 (von Heijne (1987) Nucl. Acids Res. 14:4683).Processing at this site would result in a murine B7-2 membrane boundprotein of 286 amino acids having an unmodified molecular weight ofapproximately 32 kDa. This protein would consist of an approximateextracellular Ig superfamily V and C like domains of from about aminoacid residue 24 to 246, a hydrophobic transmembrane domain of from aboutamino acid residue 247 to 265, and a long cytoplasmic domain of fromabout amino acid residue 266 to 309. The homologies to the Igsuperfamily are due to the two contiguous Ig-like domains in theextracellular region bound by the cysteines at positions 40 to 110 and157 to 216. The extracellular domain also contains nine potentialN-linked glycosylation sites and, like murine B7-1, is probablyglycosylated. Glycosylation of the murine B7-2 protein may increase themolecular weight to about 50-70 kDa. The cytoplasmic domain of murineB7-2 contains a common region which has a cysteine followed bypositively charged amino acids which presumably functions as signalingor regulatory domain within an APC. Comparison of both the nucleotideand amino acid sequences of murine B7-2 with the GenBank and EMBLdatabases yielded significant homology (about 26% amino acid sequenceidentity) with human and murine B7-1. Murine B7-2 exhibits about 50%identity and 67% similarity with its human homologue, hB7-2. E. coli(DH106/p3) transfected with a vector (plasmid pmB×4) containing a cDNAinsert encoding murine B7-2 (clone 4) was deposited with the AmericanType Culture Collection (ATCC) on Aug. 18, 1993 as Accession No. 69388.

Nucleic acids which encode novel B lymphocyte antigens from otherspecies, such as the murine B7-2, can be used to generate eithertransgenic animals or "knock out" animals which, in turn, are useful inthe development and screening of therapeutically useful reagents. Atransgenic animal (e.g., a mouse) is an animal having cells that containa transgene, which transgene was introduced into the animal or anancestor of the animal at a prenatal, e.g., an embryonic stage. Atransgene is a DNA which is integrated into the genome of a cell fromwhich a transgenic animal develops. In one embodiment, murine B7-2 cDNAor an appropriate sequence thereof can be used to clone genomic B7-2 inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain cells which express B7-2protein. Methods for generating transgenic animals, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for B7-2 transgene incorporation withtissue specific enhancers, which could result in T cell costimulationand enhanced T cell proliferation and autoimmunity. Transgenic animalsthat include a copy of a B7-2 transgene introduced into the germ line ofthe animal at an embryonic stage can be used to examine the effect ofincreased B7 expression. Such animals can be used as tester animals forreagents thought to confer protection from, for example, autoimmunedisease. In accordance with this facet of the invention, an animal istreated with the reagent and a reduced incidence of the disease,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the disease.

Alternatively, the non-human homologues of B7-2 can be used to constructa B7-2 "knock out" animal which has a defective or altered B7-2 gene asa result of homologous recombination between the endogenous B7-2 geneand altered B7-2 genomic DNA introduced into an embryonic cell of theanimal. For example, murine B7-2 cDNA can be used to clone genomic B7-2in accordance with established techniques. A portion of the genomic B7-2DNA (e.g., such as an exon which encodes an extracellular domain) can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5' and 3' ends)are included in the vector (see e.g., Thomas, K. R. and Capecchi, M. R.(1987) Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected (see e.g.,Li, E. et al. (1992) Cell 69:915). The selected cells are then injectedinto a blastocyst of an animal (e.g., a mouse) to form aggregationchimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987)pp. 113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a "knock out" animal. Progeny harbouring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for their ability to accept grafts, reject tumors anddefend against infectious diseases and can be used in the study of basicimmunobiology.

VI. Expression of B Lymphocyte Antigens

Host cells transfected to express peptides having the activity of anovel B lymphocyte antigen are also within the scope of the invention.The host cell may be any procaryotic or eucaryotic cell. For example, apeptide having B7-2 activity may be expressed in bacterial cells such asE. coli, insect cells (baculovirus), yeast, or mammalian cells such asChinese hamster ovary cells (CHO) and NS0 cells. Other suitable hostcells may be found in Goeddel, (1990) supra or are known to thoseskilled in the art.

For example, expression in eucaryotic cells such as mammalian, yeast, orinsect cells can lead to partial or complete glycosylation and/orformation of relevant inter- or intra-chain disulfide bonds ofrecombinant protein. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari. et al., (1987) Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 3:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Baculovirus vectors available for expression of proteinsin cultured insect cells (SF 9 cells) include the pAc series (Smith etal., (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M. D., (1989) Virology 170:31-39). Generally, COS cells(Gluzman, Y., (1981) Cell 23:175-182) are used in conjunction with suchvectors as pCDM8 (Seed, B., (1987) Nature 329:840) for transientamplification/expression in mammalian cells, while CHO (dhfr³¹ ChineseHamster Ovary) cells are used with vectors such as pMT2PC (Kaufman etal. (1987), EMBO J. 6:187-195) for stable amplification/expression inmammalian cells. A preferred cell line for production of recombinantprotein is the NS0 myeloma cell line available from the ECACC (catalog#85110503) and described in Galfre, G. and Milstein, C. ((1981) Methodsin Enzymology 73(13):3-46; and Preparation of Monoclonal Antibodies:Strategies and Procedures, Academic Press, New York, N.Y). Vector DNAcan be introduced into mammalian cells via conventional techniques suchas calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofectin, or electroporation.Suitable methods for transforming host cells can be found in Sambrook etal. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory press (1989)), and other laboratory textbooks. Whenused in mammalian cells, the expression vector's control functions areoften provided by viral material. For example, commonly used promotersare derived from polyoma, Adenovirus 2, cytomegalovirus and mostfrequently, Simian Virus 40.

It is known that a small faction of cells (about 1 out of 10⁵) typicallyintegrate DNA into their genomes. In order to identify these integrants,a gene that contains a selectable marker (i.e., resistance toantibiotics) is generally introduced into the host cells along with thegene of interest. Preferred selectable markers include those whichconfer resistance to drugs, such as G418, hygromycin and methotrexate.Selectable markers may be introduced on the same plasmid as the gene ofinterest or may be introduced on a separate plasmid. Cells containingthe gene of interest can be identified by drug selection; cells thathave incorporated the selectable marker gene will survive, while theother cells die. The surviving cells can then be screened for productionof novel B lymphocyte antigens by cell surface staining with ligands tothe B cell antigens (e.g., CTLA4Ig and CD28Ig). Alternatively, theprotein can be metabolically radiolabeled with a labeled amino acid andimmunoprecipitated from cell supernatant with an anti-B lymphocyteantigen monoclonal antibody or a fusion protein such as CTLA4Ig orCD28Ig.

Expression in procaryotes is most often carried out in E. coli withvectors containing constitutive or inducible promotors directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids usually to the amino terminus of the expressedtarget gene. Such fusion vectors typically serve three purposes: 1) toincrease expression of recombinant protein; 2) to increase thesolubility of the target recombinant protein; and 3) to aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the target recombinant protein to enable separation of thetarget recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-tranferase,maltose E binding protein, or protein A, respectively, to the targetrecombinant protein.

E. coli expression systems include the inducible expression vectors pTrc(Amann et al., (1988) Gene 69:301-315) and pET 11 (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 60-89; commercially available from Novagen). In thepTrc vector system, the inserted gene is expressed with a pelB signalsequence by host RNA polymerase transcription from a hybrid trp-lacfusion promoter. After induction, the recombinant protein can bepurified from the periplasmic fraction. In the pET 11 vector system, thetarget gene is expressed as non-fusion protein by transcription from theT7 gn10-lac 0 fusion promoter mediated by a coexpressed viral RNApolymerase (T7 gn1). This viral polymerase is supplied by host E. colistrains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring aT7 gn1 under the transcriptional control of the lacUV 5 promoter. Inthis system, the recombinant protein can be purified from inclusionbodies in a denatured form and, if desired, renatured by step gradientdialysis to remove denaturants.

One strategy to maximize recombinant B7-2 expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy would be to alter thenucleic acid sequence of the B7-2 gene or other DNA to be inserted intoan expression vector so that the individual codons for each amino acidwould be those preferentially utilized in highly expressed E. coliproteins (Wada et al., (1992) Nuc. Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention could be carriedout by standard DNA synthesis techniques.

Novel B lymphocyte antigens and portions thereof, expressed in mammaliancells or otherwise, can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, fractionation columnchromatography (e.g. ion exchange, gel filtration, electrophoresis,affinity chromatography, etc.) and ultimately, crystallization (seegenerally, "Enzyme Purification and Related Techniques", Methods inEnzymology, 22:233-577 (1971)). Once purified, partially or tohomogeneity, the recombinantly produced B lymphocyte antigens orportions thereof can be utilized in compositions suitable forpharmaceutical administration as described in detail herein.

VII. Modifications of Nucleic Acid and Amino Acid Sequences of theInvention and Assays for B7 Lymphocyte Antigen Activity

It will be appreciated by those skilled in the art that other nucleicacids encoding peptides having the activity of a novel B lymphocyteantigen can be isolated by the above process. Different cell lines canbe expected to yield DNA molecules having different sequences of bases.Additionally, variations may exist due to genetic polymorphisms orcell-mediated modifications of the genetic material. Furthermore, theDNA sequence of a B lymphocyte antigen can be modified by genetictechniques to produce proteins or peptides with altered amino acidsequences. Such sequences are considered within the scope of the presentinvention, where the expressed peptide is capable of either inducing orinhibiting activated T cell mediated immune responses and immunefunction.

A number of processes can be used to generate equivalents or fragmentsof an isolated DNA sequence. Small subregions or fragments of thenucleic acid encoding the B7-2 protein, for example 1-30 bases inlength, can be prepared by standard, synthetic organic chemical means.The technique is also useful for preparation of antisenseoligonucleotides and primers for use in the generation of largersynthetic fragments of B7-2 DNA.

Larger subregions or fragments of the genes encoding B lymphocyteantigens can be expressed as peptides by synthesizing the relevant pieceof DNA using the polymerase chain reaction (PCR) (Sambrook, Fritsch andManiatis, 2 Molecular Cloning; A Laboratory Manual, Cold Spring Harbor,N.Y., (1989)), and ligating the thus obtained DNA into an appropriateexpression vector. Using PCR, specific sequences of the cloned doublestranded DNA are generated, cloned into an expression vector, and thenassayed for CTLA4/CD28 binding activity. For example, to express asecreted (soluble) form of the human B7-2 protein, using PCR, a DNA canbe synthesized which does not encode the transmembrane and cytoplasmicregions of the protein. This DNA molecule can be ligated into anappropriate expression vector and introduced into a host cell such asCHO, where the B7-2 protein fragment is synthesized and secreted. TheB7-2 protein fragment can then readily be obtained from the culturemedia.

In another embodiment, mutations can be introduced into a DNA by any oneof a number of methods, including those for producing simple deletionsor insertions, systematic deletions, insertions or substitutions ofclusters of bases or substitutions of single bases, to generate variantsor modified equivalents of B lymphocyte antigen DNA. For example,changes in the human B7-2 cDNA sequence shown in FIG. 8 (SEQ ID NO:1) ormurine B7-2 cDNA sequence shown in FIG. 14 (SEQ ID NO:22) such as aminoacid substitutions or deletions are preferably obtained by site-directedmutagenesis. Site directed mutagenesis systems are well known in theart. Protocols and reagents can be obtained commercially from AmershamInternational PLC, Amersham, U.K.

Peptides having an activity of a novel B lymphocyte antigen, i.e., theability to bind to the natural ligand(s) of a B lymphocyte antigen on Tcells and either stimulate (amplify) or inhibit (block) activated T cellmediated immune responses, as evidenced by, for example, cytokineproduction and/or T cell proliferation by T cells that have received aprimary activation signal are considered within the scope of theinvention. More specifically, peptides that bind to T lymphocytes, forexample CD28⁺ cells, may be capable of delivering a costimulatory signalto the T lymphocytes, which, when transmitted in the presence of antigenand class II MHC, or other material capable of transmitting a primarysignal to the T cell, results in activation of cytokine genes within theT cell. Alternatively, such a peptide can be used in conjunction withclass I MHC to thereby activate CD8⁺ cytolytic T cells. In addition,soluble, monomeric forms of the B7-2 protein, may retain the ability tobind to their natural ligand(s) on CD28⁺ T cells but, perhaps because ofinsufficient cross-linking with the ligand, fail to deliver thesecondary signal essential for enhanced cytokine production and celldivision. Such peptides, which provide a means to induce a state ofanergy or tolerance in the cells, are also considered within the scopeof the invention.

Screening the peptides for those which retain a characteristic Blymphocyte antigen activity as described herein can be accomplishedusing one or more of several different assays. For example, the peptidescan be screened for specific reactivity with an anti-B7-2 monoclonalantibody reactive with cell surface B7-2 or with a fusion protein, suchas CTLA4Ig or CD28Ig. Specifically, appropriate cells, such as COScells, can be transfected with a B7-2 DNA encoding a peptide and thenanalyzed for cell surface phenotype by indirect immunofluorescence andflow cytometry to determine whether the peptide has B7-2 activity. Cellsurface expression of the transfected cells is evaluated using amonoclonal antibody specifically reactive with cell surface B7-2 or witha CTLA4Ig or CD28Ig fusion protein. Production of secreted forms of B7-2is evaluated using anti-B7-2 monoclonal antibody or CTLA4Ig or CD28fusion protein for immunoprecipitation.

Other, more preferred, assays take advantage of the functionalcharacteristics of the B7-2 antigen. As previously set forth, theability of T cells to synthesize cytokines depends not only on occupancyor cross-linking of the T cell receptor for antigen (the "primaryactivation signal" provided by, for example anti-CD3, or phorbol esterto produce an "activated T cell"), but also on the induction of acostimulatory signal, in this case, by interaction with a B lymphocyteantigen, such as B7-2, B7-1 or B7-3. The binding of B7-2 to its naturalligand(s) on, for example, CD28⁺ T cells, has the effect of transmittinga signal to the T cell that induces the production of increased levelsof cytokines, particularly of interleukin-2, which in turn stimulatesthe proliferation of the T lymphocytes. Other assays for B7-2 functionthus involve assaying for the synthesis of cytokines, such asinterleukin-2, interleukin-4 or other known or unknown novel cytokines,and/or assaying for T cell proliferation by CD28⁺ T cells which havereceived a primary activation signal.

In vitro, T cells can be provided with a first or primary activationsignal by anti-T3 monoclonal antibody (e.g. anti-CD3) or phorbol esteror, more preferably, by antigen in association with class II MHC. Tcells which have received a primary activation signal are referred toherein as activated T cells. B7-2 function is assayed by adding a sourceof B7-2 (e.g., cells expressing a peptide having B7-2 activity or asecreted form of B7-2) and a primary activation signal such as antigenin association with Class II MHC to a T cell culture and assaying theculture supernatant for interleukin-2, gamma interferon, or other knownor unknown cytokine. For example, any one of several conventional assaysfor interleukin-2 can be employed, such as the assay described in Proc.Natl. Acad. Sci. USA, 86:1333 (1989) the pertinent portions of which areincorporated herein by reference. A kit for an assay for the productionof interferon is also available from Genzyme Corporation (Cambridge,Mass.). T cell proliferation can also be measured as described in theExamples below. Peptides that retain the characteristics of the B7-2antigen as described herein may result in increased per cell productionof cytokines, such as IL-2, by T cells and may also result in enhanced Tcell proliferation when compared to a negative control in which acostimulatory signal is lacking.

The same basic functional assays can also be used to screen for peptideshaving B7-2 activity, but which lack the ability to deliver acostimulatory signal, but in the case of such peptides, addition of theB7-2 protein will not result in a marked increase in proliferation orcytokine secretion by the T cells. The ability of such proteins toinhibit or completely block the normal B7-2 costimulatory signal andinduce a state of anergy can be determined using subsequent attempts atstimulation of the T cells with antigen presenting cells that expresscell surface B7-2 and present antigen. If the T cells are unresponsiveto the subsequent activation attempts, as determined by IL-2 synthesisand T cell proliferation, a state of anergy has been induced. See, e.g.,Gimmi, C. D. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6586-6590; andSchwartz (1990) Science, 248, 1349-1356, for assay systems that can usedas the basis for an assay in accordance with the present invention.

It is possible to modify the structure of a peptide having the activityof a novel B lymphocyte antigen for such purposes as increasingsolubility, enhancing therapeutic or prophylactic efficacy, or stability(e.g., shelf life ex vivo and resistance to proteolytic degradation invivo). Such modified peptides are considered functional equivalents ofthe B lymphocyte antigens as defined herein. For example, a peptidehaving B7-2 activity can be modified so that it maintains the ability toco-stimulate T cell proliferation and/or produce cytokines. Thoseresidues shown to be essential to interact with the CTLA4/CD28 receptorson T cells can be modified by replacing the essential amino acid withanother, preferably similar amino acid residue (a conservativesubstitution) whose presence is shown to enhance, diminish, but noteliminate, or not effect receptor interaction. In addition, those aminoacid residues which are not essential for receptor interaction can bemodified by being replaced by another amino acid whose incorporation mayenhance, diminish, or not effect reactivity.

Another example of modification of a peptide having the activity of anovel B lymphocyte antigen is substitution of cysteine residuespreferably with alanine, serine, threonine, leucine or glutamic acidresidues to minimize dimerization via disulfide linkages. In addition,amino acid side chains of a peptide having B7-2 activity can bechemically modified. Another modification is cyclization of the peptide.

In order to enhance stability and/or reactivity, peptides having B7-2activity can be modified to incorporate one or more polymorphisms in theamino acid sequence of the antigen resulting from any natural allelicvariation. Additionally, D-amino acids, non-natural amino acids, ornon-amino acid analogs can be substituted or added to produce a modifiedprotein within the scope of this invention. Furthermore, the peptidescan be modified using polyethylene glycol (PEG) according to the methodof A. Sehon and co-workers (Wie et al., supra) to produce a peptideconjugated with PEG. In addition, PEG can be added during chemicalsynthesis of the peptide. Other modifications of the peptides includereduction/alkylation (Tarr in: Methods of Protein Microcharacterization,J. E. Silver ed., Humana Press, Clifton N.J. 155-194 (1986)); acylation(Tarr, supra); chemical coupling to an appropriate carrier (Mishell andShiigi, eds, Selected Methods in Cellular Immunology, W H Freeman, SanFrancisco, Calif. (1980), U.S. Pat. No. 4,939,239; or mild formalintreatment (Marsh (1971), Int. Arch. of Allergy and Appl. Immunol.41:199-215).

To facilitate purification and potentially increase solubility of apeptide, it is possible to add an amino acid fusion moiety to theprotein backbone. For example, hexa-histidine can be added to thepeptide for purification by immobilized metal ion affinitychromatography (Hochuli, E. et al., (1988) Bio/Technology 6:1321-1325).In addition, to facilitate isolation of a B lymphocyte antigen free ofirrelevant sequences, specific endoprotease cleavage sites can beintroduced between the sequences of a fusion moiety and the peptide. Itmay be necessary to increase the solubility of a peptide by addingfunctional groups to the peptide, or by omitting hydrophobic regions ofthe peptide.

VII. Uses of Nucleic Acid Sequences Encoding B Lymphocyte Antigens andPeptides Having B7-2 Activity

A. Molecular Probes

The nucleic acids of this invention are useful diagnostically, fortracking the progress of disease, by measuring the activation status ofB lymphocytes in biological samples or for assaying the effect of amolecule on the expresssion of a B lymphocyte antigen (e.g., detectingcellular mRNA levels). In accordance with these diagnostic assays, thenucleic acid sequences are labeled with a detectable marker, e.g., aradioactive, fluorescent, or biotinylated marker and used in aconventional dot blot or Northern hybridization procedure to probe mRNAmolecules of total or poly(A+) RNAs from a biological sample.

B. Antibody Production

The peptides and fusion proteins produced from the nucleic acidmolecules of the present invention can also be used to produceantibodies specifically reactive with B lymphocyte antigens. Forexample, by using a full-length B7-2 protein, or a peptide fragmentthereof, having an amino acid sequence based on the predicted amino acidsequence of B7-2, anti-protein/anti-peptide polyclonal antisera ormonoclonal antibodies can be made using standard methods. A mammal,(e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenicform of the protein or peptide which elicits an antibody response in themammal. The immunogen can be, for example, a recombinant B7-2 protein,or fragment thereof, a synthetic peptide fragment or a cell thatexpresses a B lymphocyte antigen on its surface. The cell can be forexample, a splenic B cell or a cell transfected with a nucleic acidencoding a B lymphocyte antigen of the invention (e.g., a B7-2 cDNA)such that the B lymphocyte antigen is expressed on the cell surface. Theimmunogen can be modified to increase its immunogenicity. For example,techniques for conferring immunogenicity on a peptide includeconjugation to carriers or other techniques well known in the art. Forexample, the peptide can be administered in the presence of adjuvant.The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassay can beused with the immunogen as antigen to assess the levels of antibodies.

Following immunization, antisera can be obtained and, if desired,polyclonal antibodies isolated from the sera. To produce monoclonalantibodies, antibody producing cells (lymphocytes) can be harvested froman immunized animal and fused with myeloma cells by standard somaticcell fusion procedures thus immortalizing these cells and yieldinghybridoma cells. Such techniques are well known in the art. For example,the hybridoma technique originally developed by Kohler and Milstein(Nature (1975) 256:495-497) as well as other techniques such as thehuman B-cell hybridoma technique (Kozbar et al., Immunol. Today (1983)4:72), the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985)(Allen R. Bliss, Inc., pages 77-96), and screening of combinatorialantibody libraries (Huse et al., Science (1989) 246:1275). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with the peptide and monoclonal antibodiesisolated.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with a peptide having theactivity of a novel B lymphocyte antigen or fusion protein as describedherein. Antibodies can be fragmented using conventional techniques andthe fragments screened for utility in the same manner as described abovefor whole antibodies. For example, F(ab')₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab')₂ fragment can betreated to reduce disulfide bridges to produce Fab' fragments. Theantibody of the present invention is further intended to includebispecific and chimeric molecules having an anti-B lymphocyte antigen(i.e., B7-2, B7-3) portion.

Particularly preferred antibodies are anti-human B7-2 monoclonalantibodies produced by hybridomas HA3.1F9, HA5.2B7 and HF2.3D1. Thepreparation and characterization of these antibodies is described indetail in Example 8. Monoclonal antibody HA3.1F9 was determined to be ofthe IgG1 isotype; monoclonal antibody HA5.2B7 was determined to be ofthe IgG2b isotype; and monoclonal antibody HF2.3D1 was determined to beof the IgG2a isotype. Hybidoma cells were deposited with the AmericanType Culture Collection, which meets the requirements of the BudapestTreaty, on Jul. 19, 1994 as ATCC Accession No. HB11688(hybridomaHA3.1F9), ATCC Accession No. HB11687(HA5.2B7) and ATCC Accession No.HB11686(HF2.3D1).

When antibodies produced in non-human subjects are used therapeuticallyin humans, they are recognized to varying degrees as foreign and animmune response may be generated in the patient. One approach forminimizing or eliminating this problem, which is preferable to generalimmunosuppression, is to produce chimeric antibody derivatives, i.e.,antibody molecules that combine a non-human animal variable region and ahuman constant region. Chimeric antibody molecules can include, forexample, the antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimeric antibodies have been described and can be used to makechimeric antibodies containing the immunoglobulin variable region whichrecognizes the gene product of the novel B lymphocyte antigens of theinvention. See, for example, Morrison et al., Proc. Natl. Acad. Sci.U.S.A. 81:6851 (1985); Takeda et al., Nature 314:452 (1985), Cabilly etal., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397;Tanaguchi et al., European Patent Publication EP171496; European PatentPublication 0173494, United Kingdom Patent GB 2177096B. It is expectedthat such chimeric antibodies would be less immunogenic in a humansubject than the corresponding non-chimeric antibody.

For human therapeutic purposes, the monoclonal or chimeric antibodiesspecifically reactive with a peptide having the activity of a Blymphocyte antigen as described herein can be further humanized byproducing human variable region chimeras, in which parts of the variableregions, especially the conserved framework regions of theantigen-binding domain, are of human origin and only the hypervariableregions are of non-human origin. General reviews of "humanized" chimericantibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207and by Oi et al. (1986) BioTechniques 4:214. Such altered immunoglobulinmolecules may be made by any of several techniques known in the art,(e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 8:7308-7312 (1983);Kozbor et al., Immunology Today, 4:7279 (1983); Olsson et al., Meth.Enzymol., 92:3-16 (1982)), and are preferably made according to theteachings of PCT Publication WO92/06193 or EP 0239400. Humanizedantibodies can be commercially produced by, for example, ScotgenLimited, 2 Holly Road, Twickenham, Middlesex, Great Britain. Suitable"humanized" antibodies can be alternatively produced by CDR or CEAsubstitution (see U.S. Pat. No. 5,225,539 to Winter; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060). Humanized antibodieswhich have reduced immunogenicity are preferred for immunotherapy inhuman subjects. Immunotherapy with a humanized antibody will likelyreduce the necessity for any concomitant immunosuppression and mayresult in increased long term effectiveness for the treatment of chronicdisease situations or situations requiring repeated antibody treatments.

As an alterntive to humanizing a monoclonal antibody from a mouse orother species, a human monoclonal antibody directed against a humanprotein can be generated. Transgenic mice carrying human antibodyrepertoires have been created which can be immunized with a human Blymphocyte antigen, such as B7-2. Splenocytes from these immunizedtransgenic mice can then be used to create hybridomas that secrete humanmonoclonal antibodies specifically reactive with a human B lymphocyteantigen (see, e.g., Wood et al. PCT publication WO 91/00906,Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. PCTpublication WO 92/03918; Kay et al. PCT publication 92/03917; Lonberg,N. et al. (1994) Nature 368:856-859; Green, L. L. et al. (1994) NatureGenet. 7:13-21; Morrison, S. L. et al. (1994) Proc. Natl. Acad. Sci. USA81:6851-6855; Bruggeman et al. (1993) Year Immunol 7:33-40; Tuaillon etal. (1993) PNAS 90:3720-3724; and Bruggeman et al. (1991) Eur J Immunol21:1323-1326).

Monoclonal antibody compositions of the invention can also be producedby other methods well known to those skilled in the art of recombinantDNA technology. An alternative method, referred to as the "combinatorialantibody display" method, has been developed to identify and isolateantibody fragments having a particular antigen specificity, and can beutilized to produce monoclonal antibodies that bind a B lymphocyteantigen of the invention (for descriptions of combinatorial antibodydisplay see e.g., Sastry et al. (1989) PNAS 86:5728; Huse et al. (1989)Science 246:1275; and Orlandi et al. (1989) PNAS 86:3833). Afterimmunizing an animal with a B lymphocyte antigen, the antibodyrepertoire of the resulting B-cell pool is cloned. Methods are generallyknown for directly obtaining the DNA sequence of the variable regions ofa diverse population of immunoglobulin molecules by using a mixture ofoligomer primers and PCR. For instance, mixed oligonucleotide primerscorresponding to the 5' leader (signal peptide) sequences and/orframework 1 (FR1) sequences, as well as primer to a conserved 3'constant region primer can be used for PCR amplification of the heavyand light chain variable regions from a number of murine antibodies(Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy canalso been used to amplify human heavy and light chain variable regionsfrom human antibodies (Larrick et al. (1991) Methods: Companion toMethods in Enzymology 2:106-110).

In an illustrative embodiment, RNA is isolated from activated B cellsof, for example, peripheral blood cells, bone marrow, or spleenpreparations, using standard protocols (e.g., U.S. Pat. No. 4,683,202;Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS (1989)8:5728-5732; and Huse et al. (1989) Science 246:1275-1281.) First-strandcDNA is synthesized using primers specific for the constant region ofthe heavy chain(s) and each of the κ and λ light chains, as well asprimers for the signal sequence. Using variable region PCR primers, thevariable regions of both heavy and light chains are amplified, eachalone or in combinantion, and ligated into appropriate vectors forfurther manipulation in generating the display packages. Oligonucleotideprimers useful in amplification protocols may be unique or degenerate orincorporate inosine at degenerate positions. Restriction endonucleaserecognition sequences may also be incorporated into the primers to allowfor the cloning of the amplified fragment into a vector in apredetermined reading frame for expression.

The V-gene library cloned from the immunization-derived antibodyrepertoire can be expressed by a population of display packages,preferably derived from filamentous phage, to form an antibody displaylibrary. Ideally, the display package comprises a system that allows thesampling of very large diverse antibody display libraries, rapid sortingafter each affinity separation round, and easy isolation of the antibodygene from purified display packages. In addition to commerciallyavailable kits for generating phage display libraries (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612), examplesof methods and reagents particularly amenable for use in generating adiverse antibody display library can be found in, for example, Ladner etal. U.S. Pat. No. 5,223,409; Kang et al. International Publication No.WO 92/18619; Dower et al. International Publication No. WO 91/17271;Winter et al. International Publication WO 92/20791; Markland et al.International Publication No. WO 92/15679; Breitling et al.International Publication WO 93/01288; McCafferty et al. InternationalPublication No. WO 92/01047; Garrard et al. International PublicationNo. WO 92/09690; Ladner et al. International Publication No. WO90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

In certain embodiments, the V region domains of heavy and light chainscan be expressed on the same polypeptide, joined by a flexible linker toform a single-chain Fv fragment, and the scFV gene subsequently clonedinto the desired expression vector or phage genome. As generallydescribed in McCafferty et al., Nature (1990) 348:552-554, completeV_(H) and V_(L) domains of an antibody, joined by a flexible (Gly₄-Ser)₃ linker can be used to produce a single chain antibody which canrender the display package separable based on antigen affinity. IsolatedscFV antibodies immunoreactive with a peptide having activity of a Blymphocyte antigen can subsequently be formulated into a pharmaceuticalpreparation for use in the subject method.

Once displayed on the surface of a display package (e.g., filamentousphage), the antibody library is screened with a B lymphocyte antigenprotein, or peptide fragment thereof, to identify and isolate packagesthat express an antibody having specificity for the B lymphocyteantigen. Nucleic acid encoding the selected antibody can be recoveredfrom the display package (e.g., from the phage genome) and subclonedinto other expression vectors by standard recombinant DNA techniques.

The antibodies of the current invention can be used therapeutically toinhibit T cell activation through blocking receptor:ligand interactionsnecessary for costimulation of the T cell. These so-called "blockingantibodies" can be identified by their ability to inhibit T cellproliferation and/or cytokine production when added to an in vitrocostimulation assay as described herein. The ability of blockingantibodies to inhibit T cell functions may result in immunosuppressionand/or tolerance when these antibodies are administered in vivo.

C. Protein Purification

The polyclonal or monoclonal antibodies of the current invention, suchas an antibody specifically reactive with a recombinant or syntheticpeptide having B7-2 activity or B7-3 activity can also be used toisolate the native B lymphocyte antigen from cells. For example,antibodies reactive with the peptide can be used to isolate thenaturally-occurring or native form of B7-2 from activated B lymphocytesby immunoaffinity chromatography. In addition, the native form of B7-3can be isolated from B cells by immunoaffinity chromatography withmonoclonal antibody BB-1.

D. Other Therapeutic Reagents

The nucleic acid sequences and novel B lymphocyte antigens describedherein can be used in the development of therapeutic reagents having theability to either upregulate (e.g., amplify) or downregulate (e.g.,suppress or tolerize) T cell mediated immune responses. For example,peptides having B7-2 activity, including soluble, monomeric forms of theB7-2 antigen or a B7-2 fusion protein, e.g., B7-2Ig, and anti-B7-2antibodies that fail to deliver a costimulatory signal to T cells thathave received a primary activation signal, can be used to block the B7-2ligand(s) on T cells and thereby provide a specific means by which tocause immunosuppression and/or induce tolerance in a subject. Suchblocking or inhibitory forms of B lymphocyte antigens and fusionproteins and blocking antibodies can be identified by their ability toinhibit T cell proliferation and/or cytokine production when added to anin vitro costimulation assay as previously described herein. In contrastto the monomeric form, stimulatory forms of B7-2, such as an intact cellsurface B7-2, retain the ability to transmit the costimulatory signal tothe T cells, resulting in an increased secretion of cytokines whencompared to activated T cells that have not received the secondarysignal.

In addition, fusion proteins comprising a first peptide having anactivity of B7-2 fused to a second peptide having an activity of anotherB lymphocyte antigen (e.g., B7-1) can be used to modify T cell mediatedimmune responses. Alternatively, two separate peptides having anactivity of B lymphocyte antigens, for example, B7-2 and B7-1, or acombination of blocking antibodies (e.g., anti-B7-2 and anti-B7-1monoclonal antibodies) can be combined as a single composition oradministered separately (simultaneously or sequentially), to upregulateor downregulate T cell mediated immune responses in a subject.Furthermore, a therapeutically active amount of one or more peptideshaving B7-2 activity and or B7-1 activity can be used in conjunctionwith other immunomodulating reagents to influence immune responses.Examples of other immunomodulating reagents include blocking antibodies,e.g., against CD28 or CTLA4, against other T cell markers or againstcytokines, fusion proteins, e.g., CTLA4Ig, or immunosuppressive drugs,e.g., cyclosporine A or FK506.

The peptides produced from the nucleic acid molecules of the presentinvention may also be useful in the construction of therapeutic agentswhich block T cell function by destruction of the T cell. For example,as described, secreted forms of a B lymphocyte antigen can beconstructed by standard genetic engineering techniques. By linking asoluble form of B7-1, B7-2 or B7-3 to a toxin such as ricin, an agentcapable of preventing T cell activation can be made. Infusion of one ora combination of immunotoxins, e.g., B7-2-ricin, B7-1 -ricin, into apatient may result in the death of T cells, particularly of activated Tcells that express higher amounts of CD28 and CTLA4. Soluble forms ofB7-2 in a monovalent form alone may be useful in blocking B7-2 function,as described above, in which case a carrier molecule may also beemployed.

Another method of preventing the function of a B lymphocyte antigen isthrough the use of an antisense or triplex oligonucleotide. For example,an oligonucleotide complementary to the area around the B7-1, B7-2 orB7-3 translation initiation site, (e.g., for B7-1, TGGCCCATGGCTTCAGA,(SEQ ID NO:20) nucleotides 326-309 and for B7-2, GCCAAAATGGATCCCCA (SEQID NO:21)), can be synthesized. One or more antisense oligonucleotidescan be added to cell media, typically at 200 μg/ml, or administered to apatient to prevent the synthesis of B7-1, B7-2 and/or B7-3. Theantisense oligonucleotide is taken up by cells and hybridizes to theappropriate B lymphocyte antigen mRNA to prevent translation.Alternatively, an oligonucleotide which binds double-stranded DNA toform a triplex construct to prevent DNA unwinding and transcription canbe used. As a result of either, synthesis of one or more B lymphocyteantigens is blocked.

In a specific embodiment, T cells are obtained from a subject andcultured ex vivo to expand the population of T cells. In a furtherembodiment the T cells are then administered to a subject. T cells canbe stimulated to proliferate in vitro by, for example, providing to theT cells a primary activation signal and a costimulatory signal, asdescribed in detail in the Examples section. A preferred costimulatorymolecule for stimulating proliferation of activated T cells, such as Tcells stimulated through their T cell receptor, is aB7-2VIg fusionprotein. However, other forms of B7-2Ig fusion proteins can also be usedto costimulate proliferation of T cells. In a specific embodiment of theinvention, activated T cells are costimulated with a B7-2VIg protein,such that a response by the activated T cells is stimulated. In oneembodiment T cells are cultured ex vivo according to the methoddescribed in PCT Application No. WO 94/29436, using B7-2Ig, or morepreferably B7-2VIg as the costimulatory molecule. The costimulatorymolecule can be soluble, attached to acell membrane or attached to asolid surface, such as a bead. In a preferred embodiment, a Thelper-type 2 (Th2) response is preferentially stimulated.

E. Therapeutic Uses by Downregulation of Immune Responses

Given the structure and function of the novel B lymphocyte antigensdisclosed herein, it is possible to downregulate the function of a Blymphocyte antigen, and thereby downregulate immune responses, in anumber of ways. Downregulation may be in the form of inhibiting orblocking an immune response already in progress or may involvepreventing the induction of an immune response. The functions ofactivated T cells may be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processwhich requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing non-responsiveness or anergyin T cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon reexposure to specific antigen in theabsence of the tolerizing agent.

Downregulating or preventing one or more B lymphocyte antigen functions,e.g., preventing high level lymphokine synthesis by activated T cells,will be useful in situations of tissue, skin and organ transplantationand in graft-versus-host disease (GVHD). For example, blockage of T cellfunction should result in reduced tissue destruction in tissuetransplantation. Typically, in tissue transplants, rejection of thetransplant is initiated through its recognition as foreign by T cells,followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a peptide having B7-2 activity alone orin conjunction with a monomeric form of a peptide having an activity ofanother B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody),prior to transplantation can lead to the binding of the molecule to thenatural ligand(s) on the immune cells without transmitting thecorresponding costimulatory signal. Blocking B lymphocyte antigenfunction in this manner prevents cytokine synthesis by immune cells,such as T cells, and thus acts as an immunosuppressant. Moreover, thelack of costimulation may also be sufficient to anergize the T cells,thereby inducing tolerance in a subject. Induction of long-termtolerance by B lymphocyte antigen-blocking reagents may avoid thenecessity of repeated administration of these blocking reagents. Toacheive sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens. For example, it may be desirable to block the function of B7-2and B7-1, B7-2 and B7-3, B7-1 and B7-3 or B7-2, B7-1 and B7-3 byadministering a soluble form of a combination of peptides having anactivity of each of these antigens or a blocking antibody (separately ortogether in a single composition) prior to transplantation.Alternatively, inhibitory forms of B lymphocyte antigens can be usedwith other suppressive agents such as blocking antibodies against otherT cell markers or against cytokines, other fusion proteins, e.g.,CTLA4Ig, or immunosuppressive drugs.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. The functionally important aspectsof B7-1 are conserved structurally between species and it is thereforelikely that other B lymphocyte antigens can function across species,thereby allowing use of reagents composed of human proteins in animalsystems. Examples of appropriate systems which can be used includeallogeneic cardiac grafts in rats and xenogeneic pancreatic islet cellgrafts in mice, both of which have been used to examine theimmunosuppressive effects of CTLA4Ig fusion proteins in vivo asdescribed in Lenschow et al., Science, 257: 789-792 (1992) and Turka etal., Proc. Natl. Acad. Sci. USA, 89: 11102-11105 (1992). In addition,murine models of GVHD (see Paul ed., Fundamental Immunology, RavenPress, New York, 1989, pp. 846-847) can be used to determine the effectof blocking B lymphocyte antigen function in vivo on the development ofthat disease.

Blocking B lymphocyte antigen function, e.g., by use of a peptide havingB7-2 activity alone or in combination with a peptide having B7-1activity and/or a peptide having B7-3 activity, may also betherapeutically useful for treating autoimmune diseases. Many autoimmunedisorders are the result of inappropriate activation of T cells that arereactive against self tissue and which promote the production ofcytokines and autoantibodies involved in the pathology of the diseases.Preventing the activation of autoreactive T cells may reduce oreliminate disease symptoms. Administration of reagents which blockcostimulation of T cells by disrupting receptor:ligand interactions of Blymphocyte antigens can be used to inhibit T cell activation and preventproduction of autoantibodies or T cell-derived cytokines which may beinvolved in the disease process. Additionally, blocking reagents mayinduce antigen-specific tolerance of autoreactive T cells which couldlead to long-term relief from the disease. The efficacy of blockingreagents in preventing or alleviating autoimmune disorders can bedetermined using a number of well-characterized animal models of humanautoimmune diseases. Examples include murine experimental autoimmuneencephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZBhybrid mice, murine autoimmune collagen arthritis, diabetes mellitus inNOD mice and BB rats, and murine experimental myasthenia gravis (seePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.840-856).

The IgE antibody response in atopic allergy is highly T cell dependentand, thus, inhibition of B lymphocyte antigen induced T cell activationmay be useful therapeutically in the treatment of allergy and allergicreactions. An inhibitory form of B7-2 protein, such as a peptide havingB7-2 activity alone or in combination with a peptide having the activityof another B lymphocyte antigen, such as B7-1, can be administered to anallergic subject to inhibit T cell mediated allergic responses in thesubject. Inhibition of B lymphocyte antigen costimulation of T cells maybe accompagnied by exposure to allergen in conjunction with appropriateMHC molecules. Allergic reactions may be systemic or local in nature,depending on the route of entry of the allergen and the pattern ofdeposition of IgE on mast cells or basophils. Thus, it may be necessaryto inhibit T cell mediated allergic responses locally or systemically byproper administration of an inhibitory form of B7-2 protein.

Inhibition of T cell activation through blockage of B lymphocyte antigenfunction may also be important therapeutically in viral infections of Tcells. For example, in the acquired immune deficiency syndrome (AIDS),viral replication is stimulated by T cell activation. Blocking B7-2function could lead to a lower level of viral replication and therebyameliorate the course of AIDS. In addition, it may also be necessary toblock the function of a combination of B lymphocyte antigens i.e., B7-1,B7-2 and B7-3. Surprisingly, HTLV-I infected T cells express B7-1 andB7-2. This expression may be important in the growth of HTLV-I infectedT cells and the blockage of B7-1 function together with the function ofB7-2 and/or B7-3 may slow the growth of HTLV-I induced leukemias.Alternatively, stimulation of viral replication by T cell activation maybe induced by contact with a stimulatory form of B7-2 protein, for suchpurposes as generating retroviruses (e.g., various HIV isolates) insufficient quantities for isolatation and use.

F. Therapeutic Uses by Upregulation of Immune Responses

Upregulation of a B lymphocyte antigen function, as a means ofupregulating immune responses, may also be useful in therapy.Upregulation of immune responses may be in the form of enhancing anexisting immune response or eliciting an initial immune response. Forexample, enhancing an immune response through stimulating B lymphocyteantigen function may be useful in cases of viral infection. Viralinfections are cleared primarily by cytolytic T cells. In accordancewith the present invention, it is believed that B7-2 and thus, B7-1 andB7-3 with their natural ligand(s) on T cells may result in an increasein the cytolytic activity of at least some T cells. It is also believedthat B7-2, B7-1, and B7-3 are involved in the initial activation andgeneration of CD8+ cytotoxic T cells. The addition of a soluble peptidehaving B7-2 activity, alone, or in combination with a peptide having theactivity of another B lymphocyte antigen, in a multi-valent form, tostimulate T cell activity through the costimulation pathway would thusbe therapeutically useful in situations where more rapid or thoroughclearance of virus would be beneficial. These would include viral skindiseases such as Herpes or shingles, in which cases the multi-valentsoluble peptide having B7-2 activity or combination of such peptideand/or a peptide having B7-1 activity and/or a peptide having B7-3activity is delivered topically to the skin. In addition, systemic viraldiseases such as influenza, the common cold, and encephalitis might bealleviated by the administration of stimulatory forms of B lymphocyteantigens systemically.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide having B7-2 activity (alone or in combination with a peptidehaving B7-1 activity and/or a peptide having B7-3 activity) or togetherwith a stimulatory form of a soluble peptide having B7-2 activity (aloneor in combination with a peptide having B7-1 activity and/or a peptidehaving B7-3 activity) and reintroducing the in vitro activated T cellsinto the patient. Another method of enhancing anti-viral immuneresponses would be to isolate infected cells from a patient, transfectthem with a nucleic acid encoding a peptide having the activity of a Blymphocyte antigen as described herein such that the cells express allor a portion of a B lymphocyte antigen on their surface, e.g., B7-2 orB7-3, and reintroduce the transfected cells into the patient. Theinfected cells would now be capable of delivering a costimulatory signalto, and thereby activate, T cells in vivo.

Stimulatory forms of B lymphocyte antigens may also be usedprophylactically in vaccines against various pathogens. Immunity againsta pathogen, e.g., a virus, could be induced by vaccinating with a viralprotein along with a stimulatory form of a peptide having B7-2 activityor another peptide having the activity of B lymphocyte antigen in anappropriate adjuvant. Alternately, an expression vector which encodesgenes for both a pathogenic antigen and a peptide having the activity ofa B lymphocyte antigen, e.g., a vaccinia virus expression vectorengineered to express a nucleic acid encoding a viral protein and anucleic acid encoding a peptide having B7-2 activity as describedherein, can be used for vaccination. Presentation of B7-2 with class IMHC proteins by, for example, a cell transfected to coexpress a peptidehaving B7-2 activity and MHC class I α chain protein and β₂microglobulin may also result in activation of cytolytic CD8+ T cellsand provide immunity from viral infection. Pathogens for which vaccinesmay be useful include hepatitis B, hepatitis C, Epstein-Barr virus,cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria andschistosomiasis.

In another application, upregulation or enhancement of B lymphocyteantigen function may be useful in the induction of tumor immunity. Tumorcells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma,carcinoma) transfected with a nucleic acid encoding at least one peptidehaving the activity of a B lymphocyte antigen, such as B7-2, can beadministered to a subject to overcome tumor-specific tolerance in thesubject. If desired, the tumor cell can be transfected to express acombination of peptides having the activity of a number of B lymphocyteantigens (e.g., B7-1, B7-2, B7-3). For example, tumor cells obtainedfrom a patient can be transfected ex vivo with an expression vectordirecting the expression of a peptide having B7-2 activity alone, or inconjuction with a peptide having B7-1 activity and/or B7-3 activity. Thetransfected tumor cells are returned to the patient to result inexpression of the peptides on the surface of the transfected cell.Alternatively, gene therapy techniques can be used to target a tumorcell for transfection in vivo.

The presence of the peptide having the activity of a B lymphocyteantigen(s) on the surface of the tumor cell provides the necessarycostimulation signal to T cells to induce a T cell mediated immuneresponse against the transfected tumor cells. In addition, tumor cellswhich lack MHC class I or MHC class II molecules, or which fail toexpress sufficient amounts of MHC class I or MHC class II molecules, canbe transfected with nucleic acid encoding all or a portion of (e.g., acytoplasmic-domain truncated portion) of an MHC class I α chain proteinand β₂ microglobulin protein or an MHC class II α chain protein and anMHC class II β chain protein to thereby express MHC class I or MHC classII proteins on the cell surface. Expression of the appropriate class Ior class II MHC in conjunction with a peptide having the activity of a Blymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediatedimmune response against the transfected tumor cell. Optionally, a geneencoding an antisense construct which blocks expression of an MHC classII associated protein, such as the invariant chain, can also becotransfected with a DNA encoding a peptide having the activity of a Blymphocyte antigen to promote presentation of tumor associated antigensand induce tumor specific immunity. Expression of B7-1 by B7 negativemurine tumor cells has been shown to induce T cell mediated specificimmunity accompanied by tumor rejection and prolonged protection totumor challenge in mice (Chen, L., et al. (1992) Cell 71, 1093-1102;Townsend, S. E. and Allison, J. P. (1993) Science 259, 368-370; Baskar,S., et al. (1993) Proc. Natl. Acad. Sci. 90, 5687-5690). Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

In another aspect, a stimulatory form of one or more soluble peptideshaving an activity of a B lymphocyte antigen can be administered to atumor-bearing patient to provide a costimulatory signal to T cells inorder to induce anti-tumor immunity.

G. Administration of Therapeutic Forms of B Lymphocyte Antigens

The peptides of the invention are administered to subjects in abiologically compatible form suitable for pharmaceutical administrationin vivo to either enhance or suppress T cell mediated immune response.By "biologically compatible form suitable for administration in vivo" ismeant a form of the protein to be administered in which any toxiceffects are outweighed by the therapeutic effects of the protein. Theterm subject is intended to include living organisms in which an immuneresponse can be elicited, e.g., mammals. Examples of subjects includehumans, dogs, cats, mice, rats, and transgenic species thereof.Administration of a peptide having the activity of a novel B lymphocyteantigen as described herein can be in any pharmacological form includinga therapeutically active amount of peptide alone or in combination witha peptide having the activity of another B lymphocyte antigen and apharmaceutically acceptable carrier. Administration of a therapeuticallyactive amount of the therapeutic compositions of the present inventionis defined as an amount effective, at dosages and for periods of timenecessary to achieve the desired result. For example, a therapeuticallyactive amount of a peptide having B7-2 activity may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of peptide to elicit a desired response inthe individual. Dosage regima may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The active compound (e.g., peptide) may be administered in a convenientmanner such as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, the activecompound may be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions which mayinactivate the compound.

To administer a peptide having B7-2 activity by other than parenteraladministration, it may be necessary to coat the peptide with, orco-administer the peptide with, a material to prevent its inactivation.For example, a peptide having B7-2 activity may be administered to anindividual in an appropriate carrier, diluent or adjuvant,co-administered with enzyme inhibitors or in an appropriate carrier suchas liposomes. Pharmaceutically acceptable diluents include saline andaqueous buffer solutions. Adjuvant is used in its broadest sense andincludes any immune stimulating compound such as interferon. Adjuvantscontemplated herein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (e.g., peptide having B7-2 activity) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient (e.g., peptide) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

When the active compound is suitably protected, as described above, theprotein may be orally administered, for example, with an inert diluentor an assimilable edible carrier. As used herein "pharmaceuticallyacceptable carrier" includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

H. Identification of Cytokines Induced by Costimulation

The nucleic acid sequences encoding peptides having the activity ofnovel B lymphocyte antigens as described herein can be used to identifycytokines which are produced by T cells in response to stimulation by aform of B lymphocyte antigen, e.g., B7-2. T cells can be suboptimallystimulated in vitro with a primary activation signal, such as phorbolester, anti-CD3 antibody or preferably antigen in association with anMHC class II molecule, and given a costimulatory signal by a stimulatoryform of B7-2 antigen, for instance by a cell transfected with nucleicacid encoding a peptide having B7-2 activity and expressing the peptideon its surface or by a soluble, stimulatory form of the peptide. Knowncytokines released into the media can be identified by ELISA or by theability of an antibody which blocks the cytokine to inhibit T cellproliferation or proliferation of other cell types that is induced bythe cytokine. An IL-4 ELISA kit is available from Genzyme (CambridgeMass.), as is an IL-7 blocking antibody. Blocking antibodies againstIL-9 and IL-12 are available from Genetics Institute (Cambridge, Mass.).

An in vitro T cell costimulation assay as described above can also beused in a method for identifying novel cytokines which may be induced bycostimulation. If a particular activity induced upon costimulation,e.g., T cell proliferation, cannot be inhibited by addition of blockingantibodies to known cytokines, the activity may result from the actionof an unkown cytokine. Following costimulation, this cytokine could bepurified from the media by conventional methods and its activitymeasured by its ability to induce T cell proliferation.

To identify cytokines which prevent the induction of tolerance, an invitro T cell costimulation assay as described above can be used. In thiscase, T cells would be given the primary activation signal and contactedwith a selected cytokine, but would not be given the costimulatorysignal. After washing and resting the T cells, the cells would berechallenged with both a primary activation signal and a costimulatorysignal. If the T cells do not respond (e.g., proliferate or produceIL-2) they have become tolerized and the cytokine has not prevented theinduction of tolerance. However, if the T cells respond, induction oftolerance has been prevented by the cytokine. Those cytokines which arecapable of preventing the induction of tolerance can be targeted forblockage in vivo in conjunction with reagents which block B lymphocyteantigens as a more efficient means to induce tolerance in transplantrecipients or subjects with autoimmune diseases. For example, one couldadminister a B7-2 blocking reagent together with a cytokine blockingantibody to a subject.

I. Identification of Molecules which Inhibit Costimulation

Another application of the peptide having the activity of a novel Blymphocyte antigen of the invention (e.g., B7-2 and B7-3) is the use ofone or more of these peptides in screening assays to discover as yetundefined molecules which are inhibitors of costimulatory ligand bindingand/or of intracellular signaling through T cells followingcostimulation. For example, a solid-phase binding assay using a peptidehaving the activity of a B lymphocyte antigen, such as B7-2, could beused to identify molecules which inhibit binding of the antigen with theappropriate T cell ligand (e.g., CTLA4, CD28). In addition, an in vitroT cell costimulation assay as described above could be used to identifymolecules which interfere with intracellular signaling through the Tcells following costimulation as determined by the ability of thesemolecules to inhibit T cell proliferation and/or cytokine production(yet which do not prevent binding of B lymphocyte antigens to theirreceptors). For example, the compound cyclosporine A inhibits T cellactivation through stimulation via the T cell receptor pathway but notvia the CD28/CTLA4 pathway. Therefore, a different intracellularsignaling pathway is involved in costimulation. Molecules whichinterfere with intracellular signaling via the CD28/CTLA4 pathway may beeffective as immunosuppressive agents in vivo (similar to the effects ofcyclosporine A).

J. Identification of Molecules which Modulate B Lymphocyte AntigenExpression

The monoclonal antibodies produced using the proteins and peptides ofthe current invention can be used in a screening assay for moleculeswhich modulate the expression of B lymphocyte antigens on cells. Forexample, molecules which effect intracellular signaling which leads toinduction of B lymphocyte antigens, e.g. B7-2 or B7-3, can be identifiedby assaying expression of one or more B lymphocyte antigens on the cellsurface. Reduced immunofluorescent staining by an anti-B7-2 antibody inthe presence of the molecule would indicate that the molecule inhibitsintracellular signals. Molecules which upregulate B lymphocyte antigenexpression result in an increased immunofluorescent staining.Alternatively, the effect of a molecule on expression of a B lymphocyteantigen, such as B7-2, can be determined by detecting cellular B7-2 mRNAlevels using a B7-2 cDNA as a probe. For example, a cell which expressesa peptide having B7-2 activity can be contacted with a molecule to betested, and an increase or decrease in B7-2 mRNA levels in the celldetected by standard technique, such as Northern hybridization analysisor conventional dot blot of mRNA or total poly(A⁺)RNAs using a B7-2 cDNAprobe labeled with a detectable marker. Molecules which modulate Blymphocyte antigen expression may be useful therapeutically for eitherupregulating or downregulating immune responses alone or in conjunctionwith soluble blocking or stimulating reagents. For instance, a moleculewhich inhibits expression of B7-2 could be administered together with aB7-2 blocking reagent for immunosuppressive purposes. Molecules whichcan be tested in the above-described assays include cytokines such asIL-4, γINF, IL-10, IL-12, GM-CSF and prostagladins.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patent applications cited throughout this application arehereby incorporated by reference.

The following methodology was used in Examples 1, 2 and 3.

METHODS AND MATERIALS

A. Cells

Mononuclear cells were isolated by Ficoll-Hypaque density gradientcentrifugation from single cell suspensions of normal human spleens andwere separated into E- and E+ fractions by rosetting with sheep redblood cells (Boyd, A. W., et al. (1985) J. Immunol. 134, 1516). B cellswere purified from the E- fraction by adherence of monocytes on plasticand depletion of residual T, natural killer cells (NK) and residualmonocytes by two treatments with anti-MsIgG and anti-MsIgM coatedmagnetic beads (Advanced Magnetics, Cambridge, Mass.), using monoclonalantibodies: anti-CD4, -CD8, -CD11b, -CD14 and -CD16. CD4+ T cells wereisolated from the E+ fraction of the same spleens after adherence onplastic and depletion of NK, B cells and residual monocytes withmagnetic beads and monoclonal antibodies: anti-CD20, -CD11b, -CD8 and-CD16. CD28+ T cells were identically isolated from the E+ fractionusing anti-CD20, -CD11b, -CD14 and -CD16 monoclonal antibodies. Theefficiency of the purification was analyzed by indirectimmunofluorescence and flow cytometry using an EPICS flow cytometer(Coulter). B cell preparations were >95% CD20+, <2% CD3+, <1% CD14+.CD4+ T cell preparations were >98% CD3+, >98% CD4+, <1% CD8+, <1% CD20+,<1% CD14+. CD28+ T cell preparations were >98% CD3+, >98% CD28+, <1%CD20+, <1% CD14+.

B. Monoclonal Antibodies and Fusion Proteins

Monoclonal antibodies were used as purified Ig unless indicatedotherwise: anti-B7:133, IgM is a blocking antibody and has beenpreviously described (Freedman, A. S. et al. (1987) Immunol. 137,3260-3267); anti-B7:B1.1, IgG1 (RepliGen Corp., Cambridge, Mass.)(Nickoloff, B., et al (1993) Am. J. Pathol. 142, 1029-1040) is anon-blocking monoclonal antibody; BB-1: IgM is a blocking antibody (Dr.E. Clark, University of Washington, Seattle, Wash.) (Yokochi, T., et al.(1982) J. Immunol. 128, 823-827); anti-CD20: B1, IgG2a (Stashenko, P.,et al. (1980) J. Immunol. 125, 1678-1685); anti-B5: IgM (Freedman, A.,et al. (1985) J. Immunol. 134, 2228-2235); anti-CD8: 7PT 3F9, IgG2a;anti-CD4: 19Thy5D7, IgG2a; anti-CD11b: Mo1, IgM and anti-CD14: Mo2, IgM(Todd, R, et al. (1981) J. Immunol. 126, 1435-1442); anti-MHC class II:9-49, IgG2a (Dr R. Todd, University of Michigan, Ann Arbor) (Todd, R.I., et al. (1984) Hum Immunol. 10, 23-40; anti-CD28: 9.3, IgG2a (Dr. C.June, Naval Research Institute, Bethesda) (Hansen, J. A., et al. (1980)Immunogenetics. 10, 247-260); anti-CD16: 3G8, IgG1 (used as ascites)(Dr. J. Ritz, Dana-Farber Cancer Institute, Boston); anti-CD3: OKT3,IgG2a hybridoma was obtained from the American Type Culture Collectionand the purified monoclonal antibody was adhered on plastic plates at aconcentration of 1 μg/ml; anti-CD28 Fab fragments were generated fromthe 9.3 monoclonal antibody, by papain digestion and purification on aprotein A column, according to the manufacturer's instructions (Pierce,Rockford, Ill.). Human CTLA4 fusion protein (CTLA4Ig) and control fusionprotein (control-Ig) were prepared as previously described (Gimmi, C.D., et al. (1993) Proc. Natl. Acad. Sci USA 90:6586-6590); Boussiotis,V., et al J. Exp. Med. (accepted for publication)).

C. CHO Cell Transfection

B7-1 transfectants (CHO-B7) were prepared from the B7-1 negative chinesehamster ovary (CHO) cell line, fixed with paraformaldehyde and used aspreviously described (Gimmi, C. D., et al. Proc. Natl. Acad. Sci USA 88,6575-6579).

D. In Vitro B Cell Activation and Selection of B7+and B7- Cells

Splenic B cells were cultured at 2×10⁶ cells/ml in complete culturemedia, {RPMI 1640 with 10% heat inactivated fetal calf serum (FCS), 2 mMglutamine, 1 mM sodium pyruvate, penicillin (100 units/ml), streptomycinsulfate (100 μg/ml) and gentamycin sulfate (5 μg/ml)}, in tissue cultureflasks and were activated by crosslinking of sIg with affinity purifiedrabbit anti-human IgM coupled to Affi-Gel 702 beads (Bio-Rad), Richmond,Calif.) (Boyd, A. W., et al., (1985) J. Immunol. 134,1516) or bycrosslinking of MHC class II with 9-49 antibody coupled to Affi-Gel 702beads. B cells activated for 72 hours, were used as total activated Bcell populations or were indirectly stained with anti-B7 (B1.1)monoclonal antibody and fluorscein isothiocyanate (FITC) labeled goatanti-mouse immunoglobulin (Fisher, Pittsburgh, Pa.), and fractionatedinto B7-1+ and B7-1- populations by flow cytometric cell sorting (EPICSElite flow cytometer, Coulter).

E. Immunoflouorescence and Flow Cytometry

For surface phenotype analysis populations of B cells activated byeither sIg or MHC class II crosslinking for 6, 12, 24, 48, 72 and 96hours were stained with either anti-B7 (133), BB-1 monoclonalantibodies, control IgM antibody, CTLA4Ig or control-Ig. Cellsuspensions were stained by two step indirect membrane staining with 10μg/ml of primary monoclonal antibody followed by the appropriatesecondary reagents. Specifically, immunoreactivity with anti-B7 (133)and BB-1 monoclonal antibodies was studied by indirect staining usinggoat anti-mouse Ig or immunoglobulin FITC (Fisher) as secondary reagentand immunoreactivity with fusion proteins was studied using biotinylatedCTLA4Ig or biotinylated control-Ig and streptavidin-phycoerythrin assecondary reagent. PBS containing 10% AB serum was used as diluent andwash media. Cells were fixed with 0.1% paraformaldehyde and analyzed ona flow cytometer (EPICS Elite Coulter).

F. Proliferation Assay

T cells were cultured at a concentration of 1×10⁵ cells per well in96-well flat bottom microtiter plate at 37° C. for 3 days in 5% CO₂.Syngeneic activated B cells (total B cell population or B7+ and B7-fractions) were irradiated (2500 rad) and added into the cultures at aconcentration of 1×10⁵ cells per well. Factors under study were added tothe required concentration for a total final volume of 200 μl per well.When indicated, T cells were incubated with anti-CD28 Fab (finalconcentration of 10 μg/ml), for 30 minutes at 4° C., prior to additionin experimental plates. Similarly, CHO-B7 or B cells were incubated withCTLA4Ig or control-Ig (10 μg/ml) for 30 minutes at 4° C. Thymidineincorporation as an index of mitogenic activity, was assessed afterincubation with 1 μCi (37 kBq) of {methyl-³ H} thymidine (Du Pont,Boston, Mass.) for the last 15 hours of the culture. The cells wereharvested onto filters and the radioactivity on the dried filters wasmeasured in a Pharmacia beta plate liquid scintilation counter.

G. IL-2 and IL-4 Assay

IL-2 and IL-4 concentrations were assayed by ELISA (R&D Systems,Minneapolis, Minn. and BioSource, Camarillo, Calif.) in culturesupernatants collected at 24 hours after initiation of the culture.

EXAMPLE 1 Expression of a Novel CTLA4 Ligand on Activated B Cells WhichInduces T Cell Proliferation

Since crosslinking surface Ig induces human resting B cells to expressB7-1 maximally (50-80%) at 72 hours, the ability of activated human Blymphocytes to induce submitogenically activated T cells to proliferateand secrete IL-2 was determined. FIG. 1 depicts the costimulatoryresponse of human splenic CD28+ T cells, submitogenically activated withanti-CD3 monoclonal antibody, to either B7 (B7-1) transfected CHO cells(CHO-B7) or syngeneic splenic B cells activated with anti-Ig for 72hours. ³ H-Thymidine incorporation was assessed for the last 15 hours ofa 72 hours culture. IL-2 was assessed by ELISA in supernatants after 24hours of culture (Detection limits of the assay: 31-2000 pg/ml). FIG. 1is representative of seventeen experiments.

Submitogenically activated CD28+ T cells proliferated and secreted highlevels of IL-2 in response to B7-1 costimulation provided by CHO-B7(FIG. 1, panel a). Both proliferation and IL-2 secretion were totallyinhibited by blocking the B7-1 molecule on CHO cells with eitheranti-B7-1 monoclonal antibody or by a fusion protein for its highaffinity receptor, CTLA4. Similarly, proliferation and IL-2 secretionwere abrogated by blocking B7-1 signalling via CD28 with Fab anti-CD28monoclonal antibody. Control monoclonal antibody or control fusionprotein had no effect. Nearly identical costimulation of proliferationand IL-2 secretion was provided by splenic B cells activated withanti-Ig for 72 hours (panel b). Though anti-B7-1 monoclonal antibodycould completely abrogate both proliferation and IL-2 secretiondelivered by CHO-B7, anti-B7-1 monoclonal antibody consistentlyinhibited proliferation induced by activated B cells by only 50% whereasIL-2 secretion was totally inhibited. In contrast to the partialblockage of proliferation induced by anti-B7-1 monoclonal antibody, bothCTLA4Ig and Fab anti-CD28 monoclonal antibody completely blockedproliferation and IL-2 secretion. These results are consistent with thehypothesis that activated human B cells express one or more additionalCTLA4/CD28 ligands which can induce T cell proliferation and IL-2secretion.

EXAMPLE 2 Activated Human Splenic B Cells Express CTLA4 Ligand(s)Distinct from B7-1

In light of the above observations, whether other CTLA4 bindingcounter-receptors were expressed on activated B cells was determined. Tothis end, human splenic B cells were activated for 72 hours with anti-Igand then stained with an anti-B7-1 monoclonal antibody (B1.1) which doesnot inhibit B7-1 mediated costimulation. Fluoroscein isothiocyanate(FITC) and mAb B1.1 were used with flow cytometric cell sorting toisolate B7-1⁺ and B7-1⁻ fractions. The resulting post-sort positivepopulation was 99% B7-1⁺ and the post-sort negative population was 98%B7-1⁻ (FIG. 2).

To examine the costimulatory potential of each population, human splenicCD28+ T cells were submitogenically stimulated with anti-CD3 monoclonalantibody in the presence of irradiated B7-1+ or B7-1- anti-Ig activated(72 hours) splenic B cells. ³ H-Thymidine incorporation was assessed forthe last 15 hours of a 72 hours culture. IL-2 was assessed by ELISA insupernatants after 24 hours of culture (Detection limits of the assay:31-2000 pg/ml). The results of FIG. 3 are representative of tenexperiments. B7-1+ B cells induced anti-CD3 activated T cells toproliferate and secrete IL-2 (FIG. 3a) but not IL-4. As was observedwith the unfractionated activated B cell population, anti-B7-1monoclonal antibody (133) inhibited proliferation only 50% butconsistently abrogated IL-2 secretion. As above, CTLA4Ig binding orblockade of CD28 with Fab anti-CD28 monoclonal antibody completelyinhibited both proliferation and IL-2 secretion. Control monoclonalantibody and control-Ig were not inhibitory. In an attempt to identifyother potential CTLA4/CD28 binding costimulatory ligand(s) which mightaccount for the residual, non-B7 mediated proliferation delivered by B7+B cells, the effect of BB-1 monoclonal antibody on proliferation andIL-2 secretion was examined. As seen, BB-1 monoclonal antibodycompletely inhibited both proliferation and IL-2 secretion (FIG. 3a).FIG. 3b displays the costimulatory potential of B7-1- activated humansplenic B cells. Irradiated B7-1- activated (72 hr) B cells could alsodeliver a significant costimulatory signal to submitogenically activatedCD4+ lymphocytes. This costimulation was not accompanied by detectableIL-2 (FIG. 3b) or IL-4 accumulation and anti-B7-1 monoclonal antibodydid not inhibit proliferation. However, CTLA4Ig, Fab anti-CD28monoclonal antibody, and BB-1 monoclonal antibody all completelyinhibited proliferation.

Phenotypic analysis of the B7-1+ and B7-1- activated splenic B cellsconfirmed the above functional results. FIG. 4 shows the cell surfaceexpression of B7-1, B7-2 and B7-3 on fractionated B7-1⁺ and B7-1⁻activated B cell. As seen in FIG. 4, B7-1+ activated splenic B cellsstained with anti-B7-1 (133) monoclonal antibody, BB-1 monoclonalantibody, and bound CTLA4-Ig. In contrast, B7- activated splenic B cellsdid not stain with anti-B7-1 (133) monoclonal antibody but did stainwith BB-1 monoclonal antibody and CTLA4Ig. These phenotypic andfunctional results demonstrate that both B7-1+ and B7-1- activated (72hours) human B lymphocytes express CTLA4 binding counter-receptor(s)which: 1) can induce submitogenically activated T cells to proliferatewithout detectable IL-2 secretion; and 2) are identified by the BB-1monoclonal antibody but not anti-B7-1 monoclonal antibody. Thus, theseCTLA4/CD28 ligands can be distinguished on the basis of their temporalexpression after B cell activation and their reactivity with CTLA4Ig andanti-B7 monoclonal antibodies. The results of FIG. 4 are representativeof five experiments.

EXAMPLE 3 Three Distinct CTLA4/CD28 Ligands Are Expressed FollowingHuman B CellActivation

To determine the sequential expression of CTLA4 bindingcounter-receptors following activation, human splenic B cells wereactivated by crosslinking of either surface Ig or MHC class II and theexpression of B7-1, B7-3 and B7-2 binding proteins were examined by flowcytometric analysis. Ig or MHC class II crosslinking induced a similarpattern of CTLA4Ig binding (FIGS. 5 and 6). FIG. 5 is representative ofthe results of 25 experiments for anti-B7-1 and BB-1 binding and 5experiments for CTLA4Ig binding. FIG. 6 is representative of 25experiments for anti-B7-1 binding and 5 experiments for CTLA4Ig binding.The results of these experiments indictes that prior to 24 hours, noneof these molecules are expressed. At 24 hours post-activation, themajority of cells express a protein that binds CTLA4Ig (B7-2), however,fewer than 20% express either B7-1 or B7-3. Crosslinking of MHC class IIinduces maximal expression and intensity of B7-1 and B7-3 at 48 hourswhereas crosslinking of Ig induces maximal expression at 72 hours andexpression declines thereafter. These results suggest that an additionalCTLA4 binding counter-receptor is expressed by 24 hours and that thetemporal expression of the distinct B7-1 and B7-3 proteins appears tocoincide.

A series of experiments was conducted to determine whether the temporalexpression of CTLA4 binding counter-receptors differentially correlatedwith their ability to costimulate T cell proliferation and/or IL-2secretion. Human splenic CD28+T cells submitogenically stimulated withanti-CD3 were cultured for 72 hours in the presence of irradiated humansplenic B cells that had been previously activated in vitro by sIgcrosslinking for 24, 48, or 72 hours. IL-2 secretion was assessed byELISA in supernatants after 24 hours and T cell proliferation asassessed by ³ H-thymidine incorporation for the last 15 hours of a 72hour culture. The results of FIG. 7 are representative of 5 experiments.As seen in FIG. 7a, 24 hour activated B cells provided a costimulatorysignal which was accompanied by modest levels of IL-2 production,although the magnitude of proliferation was significantly less thanobserved with 48 and 72 hours activated human B cells (note differencesin scale for ³ H-Thymidine incorporation). Neither proliferation norIL-2 accumulation was inhibited by anti-B7-1 (133) or BB-1. In contrast,with CTLA4Ig and anti-CD28 Fab monoclonal antibody totally abrogatedproliferation and IL-2 accumulation. B cells activated for 48 hours,provided costimulation which resulted in nearly maximal proliferationand IL-2 secretion (FIG. 7b). Here, anti-B7-1 (133) monoclonal antibody,inhibited proliferation approximately 50% but totally blocked IL-2accumulation. BB-1 monoclonal antibody totally inhibited bothproliferation and IL-2 secretion. As above, CTLA4Ig and Fab anti-CD28also totally blocked proliferation and IL-2 production. Finally, 72 houractivated B cells induced T cell response identical to that induced by48 hour activated B cells. Similar results are observed if thesubmitogenic signal is delivered by phorbol myristic acid (PMA) and ifthe human splenic B cells are activated by MHC class II rather than Igcrosslinking. These results indicate that there are three CTLA4 bindingmolecules that are temporarily expressed on activated B cells and eachcan induce submitogenically stimulated T cells to proliferate. Two ofthese molecules, the early CTLA4 binding counter-receptor (B7-2) andB7-1 (133) induce IL-2 production whereas B7-3 induces proliferationwithout detectable IL-2 production.

Previous studies provided conflicting evidence whether the anti-B7monoclonal antibody,133 and monoclonal antibody BB-1 identified the samemolecule (Freedman, A. S. et al. (1987) Immunol. 137, 3260-3267;Yokochi, T., et al. (1982) J. Immunol. 128, 823-827; Freeman, G. J., etal. (1989) J. Immunol. 143, 2714-2722.). Although both monoclonalantibodies identified molecules expressed 48 hours following humanB-cell activation, several reports suggested that B7 (B7-1) and themolecule identified by monoclonal antibody BB-1 were distinct since theywere differentially expressed on cell lines and B cell neoplasms(Freedman, A. S. et al. (1987) Immunol. 137, 3260-3267; Yokochi, T., etal. (1982) J. Immunol. 128, 823-827; Freeman, G. J., et al. (1989) J.Immunol. 143, 2714-2722; Clark, E and Yokochi, T. (1984) LeukocyteTyping, 1st International References Workshop. 339-346; Clark, E., etal. (1984) Leukocyte Typing, 1st International References Workshop.740). In addition, immunoprecipitation and Western Blotting with theseIgM monoclonal antibodies suggested that they identified differentmolecules (Clark, E and Yokochi, T. (1984) Leukocyte Typing, 1stInternational References Workshop. 339-346; Clark, E., et al. (1984)Leukocyte Typing, 1st International References Workshop. 740). Theoriginal anti-B7 monoclonal antibody, 133, was generated by immunizationwith anti-immunoglobulin activated human B lymphocytes whereas the BB-1monoclonal antibody was generated by immunization with a baboon cellline. Thus, the BB-1 monoclonal antibody must identify an epitope onhuman cells that is conserved between baboons and humans.

Following the molecular cloning and expression of the human B7 gene(B7-1), B7 transfected COS cells were found to be identically stainedwith the anti-B7 (133) and BB-1 monoclonal antibodies and that they bothprecipitated the identical broad molecular band (44-54 kD) stronglysuggesting that they identified the same molecule (Freeman, G. J., etal. (1989) J. Immunol. 143, 2714-2722). This observation was unexpectedsince the gene encoding the molecule identified by the BB-1 monoclonalantibody had been previously mapped to chromosome 12 (Katz, F. E., etal. (1985) Eur. J. Immunol. 103-6), whereas the B7 gene was located bytwo groups on chromosome 3 (Freeman, G. J., et al. (1992) Blood. 79,489-494; Selvakumar, A., et al. (1992) Immunogenetics 36, 175-181.).Subsequently, additional discrepancies between the phenotypic expressionof B7 (B7-1) and the molecule identified by the BB-1 monclonal antibodywere noted. BB-1 monoclonal antibody stained thymic epithelial cells(Turka, L. A., et al. (1991) J. Immunol. 146, 1428-36; Munro, J. M., etal. Blood submitted.) and keratinocytes (Nickoloff, B., et al (1993) Am.J. Pathol. 142, 1029-1040; Augustin, M., et al. (1993) J. Invest.Dermatol. 100, 275-281.) whereas anti-B7 did not. Recently, Nickoloffetal. (1993) Am. J. Pathol. 142, 1029-1040, reported discordant expressionof the molecule identified by the BB-1 monoclonal antibody and B7 onkeratinocytes using a BB-1 and anti-B7 (B1.1 and 133) monoclonalantibodies. Nickoloffet al. also demonstrated that these BB-1 positivecells did not express B7 mRNA yet bound CD28 transfected COS cellsproviding further support for the existence of a distinct protein whichbinds monoclonal antibody BB-1.

The present findings confirm that there is an additional CTLA4counter-receptor identified by the BB-1 monoclonal antibody, B7-3, andthat this protein appears to be functionally distinct from B7-1 (133).Although the expression of B7-1 and B7-3 following B cell activationappears to be concordant on B7 positive B cells, these studiesdemonstrate that the B7-3 molecule is also expressed on B7 negativeactivated B cells. More importantly, the B7-3 molecule appears to becapable of inducing T cell proliferation without detectable IL-2 or IL-4production. This result is similar to the previous observation thatICAM-1 could costimulate T cell proliferation without detectable IL-2 orIL-4 production (Boussiotis, V., et al J. Exp. Med. (accepted forpublication)). These data indicate that the BB-1 monoclonal antibodyrecognizes an epitope on the B7-1 protein and that this epitope is alsofound on a distinct B7-3 protein, which also has costimulatory function.Phenotypic and blocking studies demonstrate that the BB-1 monoclonalantibody could detect one (on B7 negative cells) or both (on B7 positivecells) of these proteins. In contrast, the anti-B7 monoclonalantibodies, 133 and B1.1 detect only the B7-1 protein. Taken together,these results suggest that by 48 hours post B-cell activation bycrosslinking of surface immunoglobulin or MHC class II, B cells expressat least two distinct CTLA4 binding counter-receptors, one identified byboth anti-B7 and BB-1 monclonal antibodies and the other identified onlyby BB-1 monoclonal antibody.

The B7-2 antigen is not detectable on activated B cells after 12 hours,but by 24 hours it is strongly expressed and functional. This moleculeappears to signal via CD28 since proliferation and IL-2 production arecompletely blocked by Fab anti-CD28 monoclonal antibody. At 48 hourspost activation, IL-2 secretion seems to be accounted for by B7-1costimulation, since anti-B7 monoclonal antibody completely inhibitsIL-2 production.

Previous studies and results presented here demonstrate that B7 (B7-1)is neither expressed (Freedman, A. S. et al. (1987) Immunol. 137,3260-3267; Freedman, A. S., et al. (1991) Cell. Immunol. 137, 429-437)nor capable of costimulating T cell proliferation or IL-2 secretionuntil 48 hours post B-cell activation. Previous studies have shown thatactivation of T cells via the TCR in the absence of costimulation(Gimmi, C. D., et al. (1993) Proc. Natl. Acad. Sci USA 90:6586-6590;Schwartz, R. H., et al. (1989) Cold Spring Harb. Symp. Quant. Biol 54,605-10; Beverly, B., et al. (1992) Int. Immunol. 4, 661-671.) and lackof IL-2 (Boussiotis, V., et al J. Exp. Med. (submitted); Beverly, B., etal. (1992) Int. Immunol. 4, 661-671; Wood, M., et al. (1993) J. Exp.Med. 177, 597-603) results in anergy. If B7-1 were the onlycostimulatory molecule capable of inducing IL-2 secretion, T cells wouldbe anergized within the first 24 hours following activation since thereis no B7-1 present to costimulate IL-2 production. Therefore, theexistence of another, early inducible costimulatory molecule, which cancostimulate IL-2 secretion during the first 24 hours would be necessaryto induce an effective immune response rather than anergy. Theappearance of the early CTLA4 binding counter-receptor, B7-2, between 12and 24 hours post B cell activation, fulfills this function.

Two observations shed light on the biologic and potential clinicalsignificance of these two additional CTLA4 binding counter-receptors.First, B7 (B7-1) deficient mouse has been developed and its antigenpresenting cells were found to still bind CTLA4Ig (Freeman and Sharpemanuscript in preparation). This mouse is viable and isolatedmononuclear cells induce detectable levels of IL-2 when cultured with Tcells in vitro. Therefore, an alternative CD28 costimulatorycounter-receptor or an alternative IL-2 producing pathway must befunctional. Second, thus far the most effective reagents to induceantigen specific anergy in murine and human systems are CTLA4Ig and Fabanti-CD28, whereas anti-B7 monoclonal antibodies have been much lesseffective (Harding, F. A., et al. (1992) Nature. 356, 607-609; Lenschow,D. J., et al. (1992) Science. 251, 789-792; Chen, L., et al. (1992)Cell. 71, 1093-1102; Tan, P., et al. (1993) J. Exp. Med. 177, 165-173.).These observations are also consistent with the hypothesis thatalternative CTLA4/CD28 ligands capable of inducing IL-2 exist, and takentogether with the results presented herein, suggest that all three CTLA4binding counter-receptors may be critical for the induction of T cellimmunity. Furthermore, their blockade will likely be required for theinduction of T cell anergy. Identical results have been observed in themurine system with the identification of two CTLA4 binding ligands,corresponding to the human B7-1 and B7-2 molecules. APCs in the B7deficient mouse bind to the CTLA4 and can induce IL-2 secretion. Takentogether, these observations indicate that multiple CTLA-4 bindingcounter-receptors exist and sequentially costimulate T cell activationin the murine system.

EXAMPLE 4 Cloning, Sequencing and Expression of the B7-2 Antigen

A. Construction of cDNA Library

A cDNA library was constructed in the pCDM8 vector (Seed, Nature,329:840 (1987)) using poly (A)⁺ RNA from the human anti-IgM activated Bcells as described (Aruffo et al, Proc. Natl. Acad. Sci. USA, 84:3365(1987)). Splenic B cells were cultured at 2×10⁶ cells/ml in completeculture media, {RPMI 1640 with 10% heat inactivated fetal calf serum(FCS), 2 mM glutamine, 1 mM sodium pyruvate, penicillin (100 units/ml),streptomycin sulfate (100 μg/ml) and gentamycin sulfate (5 μg/ml)}, intissue culture flasks and were activated by crosslinking of sIg withaffinity purified rabbit anti-human IgM coupled to Affi-Gel 702 beads(Bio-Rad), Richmond, Calif.) (Boyd, A. W., et al., (1985) J Immunol.134,1516). Activated B cells were harvested after 1/6, 1/2, 4, 8 12, 24,48, 72 and 96 hours.

RNA was prepared by homogenizing activated B cells in a solution of 4Mguanidine thiocyanate, 0.5% sarkosyl, 25 mM EDTA, pH 7.5, 0.13% Sigmaanti-foam A, and 0.7% mercaptoethanol. RNA was purified from thehomogenate by centrifugation for 24 hour at 32,000 rpm through asolution of 5.7M CsCl, 10 mM EDTA, 25 mM Na acetate, pH 7. The pellet ofRNA was dissolved in 5% sarkosyl, 1 mM EDTA, 10 mM Tris, pH 7.5 andextracted with two volumes of 50% phenol, 49% chloroform, 1% isoamylalcohol. RNA was ethanol precipitated twice. Poly (A)⁺ RNA used in cDNAlibrary construction was purified by two cycles of oligo (dT)-celluloseselection.

Complementary DNA was synthesized from 5.5 μg of anti-IgM activatedhuman B cell poly(A)⁺ RNA in a reaction containing 50 mM Tris, pH 8.3,75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 500 μM dATP, dCTP, dGTP,dTTP, 50 μg/ml oligo(dT)₁₂₋₁₈, 180 units/ml RNasin, and 10,000 units/mlMoloney-MLV reverse transcriptase in a total volume of 55 μl at 37° for1 hr. Following reverse transcription, the cDNA was converted todouble-stranded DNA by adjusting the solution to 25 mM Tris, pH 8.3, 100mM KCl, 5 mM MgCl₂, 250 μM each dATP, dCTP, dGTP, dTTP, 5 mMdithiothreitol, 250 units/ml DNA polymerase I, 8.5 units/ml ribonucleaseH and incubating at 16° for 2 hr. EDTA was added to 18 mM and thesolution was extracted with an equal volume of 50% phenol, 49%chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes ofethanol in the presence of 2.5M ammonium acetate and with 4 microgramsof linear polyacrylamide as carrier. In addition, cDNA was synthesizedfrom 4 μg of anti-IgM activated human B cell poly(A)⁺ RNA in a reactioncontaining 50 mM Tris, pH 8.8, 50 μg/ml oligo(dT)₁₂₋₁₈, 327 units/mlRNasin, and 952 units/ml AMV reverse transcriptase in a total volume of100 μl at 42° for 0.67 hr. Following reverse transcription, the reversetranscriptase was inactivated by heating at 70° for 10 min. The cDNA wasconverted to double-stranded DNA by adding 320 μl H₂ O and 80 μl of asolution of 0.1M Tris, pH 7.5, 25 mM MgCl₂, 0.5M KCl, 250 μg/ml bovineserum albumin, and 50 mM dithiothreitol, and adjusting the solution to200 μM each dATP, dCTP, dGTP, dTTP, 50 units/ml DNA polymerase I, 8units/ml ribonuclease H and incubating at 16° C. for 2 hours. EDTA wasadded to 18 mM and the solution was extracted with an equal volume of50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA was precipitatedwith two volumes of ethanol in the presence of 2.5M ammonium acetate andwith 4 micrograms of linear polyacrylamide as carrier.

The DNA from 4 μg of AMV reverse transcription and 2 μg of Moloney MLVreverse transcription was combined. Non-selfcomplementary BstXI adaptorswere added to the DNA as follows: The double-stranded cDNA from 6 μg ofpoly(A)⁺ RNA was incubated with 3.6 μg of a kinased oligonucleotide ofthe sequence CTTTAGAGCACA (SEQ ID NO:15) and 2.4 μg of a kinasedoligonucleotide of the sequence CTCTAAAG (SEQ ID NO:16) in a solutioncontaining 6 mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovineserum albumin, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1mM spermidine, and 600 units T4 DNA ligase in a total volume of 0.45 mlat 15° C. for 16 hours. EDTA was added to 34 mM and the solution wasextracted with an equal volume of 50% phenol, 49% chloroform, 1% isoamylalcohol. DNA was precipitated with two volumes of ethanol in thepresence of 2.5M ammonium acetate.

DNA larger than 600 bp was selected as follows: The adaptored DNA wasredissolved in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCl, 0.1% sarkosyland chromatographed on a Sepharose CL-4B column in the same buffer. DNAin the void volume of the column (containing DNA greater than 600 bp)was pooled and ethanol precipitated.

The pCDM8 vector was prepared for cDNA cloning by digestion with BstXIand purification on an agarose gel. Adaptored DNA from 6 μg of poly(A)⁺RNA was ligated to 2.25 μg of BstXI cut pCDM8 in a solution containing 6mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovine serum albumin,7 mM mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1 mM spermidine,and 600 units T4 DNA ligase in a total volume of 1.5 ml at 15° for 24hr. The ligation reaction mixture was transformed into competent E. coliMC1061/P3 and a total of 4,290,000 independent cDNA clones wereobtained.

Plasmid DNA was prepared from a 500 ml culture of the originaltransformation of the cDNA library. Plasmid DNA was purified by thealkaline lysis procedure followed by twice banding in CsCl equilibriumgradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1987)).

B. Cloning Procedure

In the first round of screening, thirty 100 mm dishes of 50% confluentCOS cells were transfected with 0.05 μg/ml anti-IgM activated human Bcells library DNA using the DEAE-Dextran method (Seed et al, Proc. Natl.Acad. Sci. USA, 84:3365 (1987)). The cells were trypsinized andre-plated after 24 hours. After 47 hours, the cells were detached byincubation in PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37° C. for 30min. The detached cells were treated with 10 μg/ml/CTLA4Ig and CD28Igfor 45 minutes at 4° C. Cells were washed and distributed into panningdishes coated with affinity-purified Goat anti-human IgG antibody andallowed to attach at room temperature. After 3 hours, the plates weregently washed twice with PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide, 5% FCSand once with 0.15M NaCl, 0.01M Hepes, pH 7.4, 5% FCS. Episomal DNA wasrecovered from the panned cells and transformed into E. coli DH10B/P3.The plasmid DNA was re-introduced into COS cells via spheroplast fusionas described (Seed et al, Proc. Natl. Acad. Sci. USA, 84:3365 (1987))and the cycle of expression and panning was repeated twice. In thesecond and third rounds of selection, after 47 hours, the detached COScells were first incubated with α-B7-1 mAbs (133 and B1.1, 10 μg/ml),and COS cells expressing B7-1 were removed by α-mouse IgG and IgM coatedmagnetic beads. COS cells were then treated with 10 μg/ml of humanCTLA4Ig (hCTLA4Ig) and human CD28Ig (hCD28Ig) and human B7-2 expressingCOS cells were selected by panning on dishes with goat anti-human IgGantibody plates. After the third round, plasmid DNA was prepared fromindividual colonies and transfected into COS cells by the DEAE-Dextranmethod. Expression of B7-2 on transfected COS cells was analyzed byindirect immunofluorescence with CTLA4Ig.

After the final round of selection, plasmid DNA was prepared fromindividual colonies. A total of 4 of 48 candidate clones contained acDNA insert of approximately 1.2 kb. Plasmid DNA from these four cloneswas transfected into COS cells. All four clones were strongly positivefor B7-2 expression by indirect immunofluorescence using CTLA4Ig andflow cytometric analysis.

C. Sequencing

The B7-2 cDNA insert in clone29 was sequenced in the pCDM8 expressionvector employing the following strategy. Initial sequencing wasperformed using sequencing primers T7, CDM8R (Invitrogen) homologous topCDM8 vector sequences adjacent to the cloned B7-2 cDNA (see Table I).Sequencing was performed using dye terminator chemistry and an ABIautomated DNA sequencer. (ABI, Foster City, Calif.). DNA sequenceobtained using these primers was used to design additional sequencingprimers (see Table I). This cycle of sequencing and selection ofadditional primers was continued until the B7-2 cDNA was completelysequenced on both strands.

                                      TABLE I                                     __________________________________________________________________________    T7 (F)   (SEQ ID NO:3)                                                                           5'd[TAATACGACTCACTATAGGG]3'                                CDM8 (R) (SEQ ID NO:4)                                                                                    5'd[TAAGGTTCCTTCACAAAG]3'                         CDM8 RGV (2)                                                                           (SEQ ID NO:5)                                                                                 5'd[ACTGGTAGGTATGGAAGATCC]3'                         HBX29-5P (2R)                                                                          (SEQ ID NO:6)                                                                            5'd[ATGCGAATCATTCCTGTGGGC]3'                              HBX29-5P (2F)                                                                          (SEQ ID NO:7)                                                                            5'd[AAAGCCCACAGGAATGATTCG]3'                              HBX29-5P (SEQ ID NO:8)                                                                                   5'd[CTCTCAAAACCAAABCCTGAG]3'                       5PA      (SEQ ID NO:9)                                                                                        5'd[TTAGGTCACAGCAGAAGCAGC]3'                  5PA (3FA)                                                                              (SEQ ID NO:10)                                                                                5'd[TCTGGAAACTGACAAGACGCG]3'                         HBX29-5P (1R)                                                                          (SEQ ID NO:11)                                                                            5'd[CTCAGGCTTTGGTTTTGAGAG]3'                             HBX29-3P (1R)                                                                          (SEQ ID NO:12)                                                                            5'd[CACTCTCTTCCCTCTCCATTG]3'                             HBX29-5P (3R)                                                                          (SEQ ID NO:13)                                                                            5'd[GACAAGCTGATGGAAACGTCG]3'                             HBX29-3P (1P)                                                                          (SEQ ID NO:14)                                                                            5'd[CAATGGAGAGGGAAGAGAGTG]3'                             __________________________________________________________________________

The human B7-2 clone 29 contained an insert of 1,120 base pairs with asingle long open reading frame of 987 nucleotides and approximately 27nucleotides of 3' noncoding sequences (FIG. 8 (SEQ ID NO:1)). Thepredicted amino acid sequence encoded by the open reading frame of theprotein is shown below the nucleotide sequence in FIG. 8. The encodedprotein, human B7-2, is predicted to be 329 amino acids in length (SEQID NO:2). This protein sequence exhibits many features common to othertype 1 Ig superfamily embrane proteins. Protein translation is predictedto begin at the ATG codon (nucleotide 107-109) based on DNA homology inthis region with the consensus eukaryotic translation initiation site(Kozak, M. (1987) Nucl. Acids Res. 15:8125-8148). The amino terminus ofthe human B7-2 protein (amino acids 1 to 23) has the characteristics ofa secretory signal peptide with a predicted cleavage between thealanines at positions 23 and 24 (von Heijne (1986) Nucl. Acids Res.14:4683). Processing at this site would result in a human B7-2 membranebound protein of 306 amino acid with an unmodified molecular weight ofapproximately 34 kDa. This protein would consist of an extracellular Igsuperfamily V and C like domains, of from about amino acid residue24-245, a hydrophobic transmembrane domain of from about amino acidresidue 246-268 and a long cytoplasmic domain of from about amino acidresidue 269-329. The homologies to the Ig superfamily are due to the twocontiguous Ig-like domains in the extracellular region bound by thecysteines at positions 40 to 110 and 157 to 218. The extracellulardomain also contains eight potential N-linked glycosylation sites. E.coli transfected with a vector containing the cDNA insert of clone 29,encoding the human B7-2 protein, was deposited with the American TypeCulture Collection (ATCC) on Jul. 26, 1993 as Accession No. 69357.

Comparison of both the nucleotide and amino acid sequences of human B7-2with the GenBank and EMBL databases showed that only the human andmurine B7-1 proteins are related. Alignment of the three B7 proteinsequences (see FIG. 13) shows that human B7-2 has approximately 26%amino acid identity with human B7-1. FIG. 13 represents the comparisonof the amino acid sequences for human B7-2 (hB7-2) (SEQ ID NO:2), humanB7-1 (hB7-1) (SEQ ID NO:28 and 29) and murine B7 (mB7) (SEQ ID NO:30 and31). The amino acid sequences for the human B7-1 and murine B7 (referredto herein as murine B7-1) can be found in Genbank at Accession #M27533and X60958 respectively. Vertical lines in FIG. 13 show identical aminoacids between the hB7-2 and hB7-1 or mB7. Identical amino acids betweenhB7-1 and mB7 are not shown. The hB7-2 protein exhibits the same generalstructure as hB7-1 as defined by the common cysteines (positions 40 and110, IgV domains; positions 157 and 217, IgC domain) which the Igsuperfamily domains and by many other common amino acids. Since bothhB7-1 and mB7 have been shown to bind to both human CTLA4 and humanCD28, the amino acids in common between these two related proteins willbe those necessary to comprise a CTLA4 or CD28 binding sequence. Anexample of such a sequence would be the KYMGRTSFD (position 81-89,hB7-2) (SEQ ID NO:17) or KSQDNVTELYDVS (position 188-200, hB7-2) (SEQ IDNO:18). Additional related sequences are evident from the sequencecomparison and others can be inferred by considering homologous relatedamino acids such as aspartic acid and glutamic acid, alanine and glycineand other recognized functionally related amino acids. The B7 sequencesshare a highly positive charged domain with the cytoplasmic portionWKWKKKKRPRNSYKC (position 269-282, hB7-2) (SEQ ID NO:19) which isprobably involved in intracellular signaling.

EXAMPLE 5 Characterization of the Recombinant B7-2 Antigen

A. B7-2 Binds CTLA4Ig and Not Anti-B7-1 and Anti-B7-3 MonoclonalAntibodies

COS cells transfected with either vector DNA (pCDNAI), or an expressionplasmid containing B7-1 (B7-1) or B7-2 (B7-2) were prepared. After 72hours, the transfected COS cells were detached by incubation in PBScontaining 0.5 mM EDTA and 0.02% Na azide for 30 min. at 37° C. Cellswere analyzed for cell surface expression by indirect immunofluorescenceand flow cytometric analysis using fluoroscein isothiocyanate conjugated(FITC) goat-anti-mouse Ig or goat-anti-human IgG FITC (FIG. 9). Cellsurface expression of B7-1 was detected with mAbs 133 (anti-B7-1) andBB-1 (anti-B7-1 and anti-B7-3) and with CTLA4Ig, whereas B7-2 reactedonly with CTLA4Ig. Neither of the B7 transfectants showed any stainingwith the isotype controls (IgM or control Ig). The vector transfectedCOS cells showed no staining with any of the detection reagents. Inaddition, none of the cells showed any staining with the FITC labeleddetection reagents and alone. This demonstrates that B7-2 encodes aprotein that is a CTLA4 counter-receptor but is distinct from B7-1 andB7-3.

B. RNA Blot Analysis of B7-2 Expression in Unstimulated and ActivatedHuman B Cells, Cell Lines, and Myelomas

Human splenic B cells were isolated by removing T cells and monocytes aspreviously described (Freedman, A. S., Freeman, G. J., Horowitz, J. C.,Daley, J., Nadler, L. M., J. Immunol. (1987) 131:3260-3267). Splenic Bcells were activated using anti-Ig beads and cells were harvested at theindicated times (Freedman et al., (1987), cited supra). Human myelomasfrom bone marrow specimens were enriched by removing T cells andmonocytes using E rosettes and adherence as previously described(Freeman, G. J., et al., J. Immunol. (1989) 143:2714-2722). RNA wasprepared by guanidine thiocyanate homogenization and cesium chloridecentrifugation. Equal amounts of RNA (20 μg) were electrophoresed on anagarose gel, blotted, and hybridized to ³² P-labelled B7-2 cDNA. FIG.10, panel a, shows RNA blot analysis of unstimulated and anti-Igactivated human splenic B cells and of cell lines including Raji (B cellBurkitts lymphoma), Daudi (B cell Burkitt's lymphoma), RPMI 8226(myeloma), K562 (erythroleukemia), and REX (T cell acute lymphoblasticleukemia). FIG. 10, panel b shows RNA blot analysis of human myelomaspecimens.

Three mRNA transcripts of 1.35, 1.65 and 3.0 kb were identified byhybridization to the B7-2 cDNA (FIG. 10, panel b). RNA blot analysisdemonstrated that B7-2 mRNA is expressed in unstimulated human splenic Bcells and increases 4-fold following activation (FIG. 10, panel a). B7-2mRNA was expressed in B cell neoplastic lines (Raji, Daudi) and amyeloma (RPMI 8226) but not in the erythroleukemia K562 and the T cellline REX. In contrast, we have previously shown that B7-1 mRNA is notexpressed in resting B cells and is transiently expressed followingactivation (G. J. Freeman et al. (1989) supra). Examination of mRNAisolated from human myelomas demonstrates that B7-2 mRNA is expressed in6 of 6 patients, whereas B7-1 was found in only 1 of these 6 (G. J.Freeman et al. (1989) supra). Thus, B7-1 and B7-2 expression appears tobe independently regulated.

C. Costimulation

Human CD28⁺ T cells were isolated by immunomagnetic bead depletion usingmonoclonal antibodies directed against B cells, natural killer cells andmacrophages as previously described (Gimmi, C. D., et al. (1993) Proc.Natl. Acad. Sci. USA 90, 6586-6590). B7-1, B7-2 and vector transfectedCOS cells were harvested 72 hours after transfection, incubated with 25μg/ml of mitomycin-C for 1 hour, and then extensively washed. 10⁵ CD28⁺and T cells were incubated with 1 ng/ml of phorbol myristic acid (PMA)and the indicated number of COS transfectants (FIG. 11). As shown inFIG. 11, panel a, T cell proliferation was measured by 3H-thymidine (1μCi) incorporated for the last 12 hours of a 72 hour incubation. Panel bof FIG. 11 shows IL-2 production by T cells as measured by ELISA(Biosource, Calif.) using supernatants harvested 24 hours after theinitiation of culture.

D. B7-2 Costimulation is not Blocked by Anti-B7-1 and Anti-B7-3 mAbs butis Blocked by CTLA4-Ig and Anti-CD28 Fab

Human CD28⁺ T cells were isolated by immunomagnetic bead depletion usingmAbs directed against B cells, natural killer cells, and macrophages aspreviously described (Gimnmi, C. D., Freeman, G. J., Gribben, J. G.,Gray, G., Nadler, L. M. (1993) Proc. Natl. Acad. Sci USA 90, 6586-6590).B7-1, B7-2, and vector transfected COS cells were harvested 72 hoursafter transfection, incubated with 25 μg/ml of mitomycin-C for 1 hour,and then extensively washed. 10⁵ CD28⁺ T cells were incubated with 1ng/ml of phorbol myristic acetate (PMA) and 2×10⁴ COS transfectants.Blocking agents (10 μg/ml) are indicated on the left side of FIG. 12 andinclude: 1) no monoclonal antibody (no blocking agents), 2) mAb 133(anti-B7-1 mAb), 3) mAb BB1 (anti-B7-1 and anti-B7-3 mAb), 4) mAb B5(control IgM mAb), 5) anti-CD28 Fab (mnAb 9.3), 6) CTLA-Ig, and 7)control Ig. Panel a of FIG. 11 shows proliferation measured by ³H-thymidine (1 μCi) incorporation for the last 12 hours of a 72 hourincubation. FIG. 11, panel b, shows IL-2 production as measured by ELISA(Biosource, Calif.) using supernatants harvested 24 hours after theinitiation of culture.

B7-1 and B7-2 transfected COS cells costimulated equivalent levels of Tcell proliferation when tested at various stimulator to responder ratios(FIG. 11). Like B7-1, B7-2 transfected COS cell costimulation resultedin the production of IL-2 over a wide range of stimulator to respondercell ratios (FIG. 11). In contrast, vector transfected COS cells did notcostimulate T cell proliferation or IL-2 production.

E. B7-2 Costimulation is not Blocked by Anti-B7-1 and Anti-B7-3 mAbs butis Blocked by CTLA4-Ig and Anti-CD28 Fab

Human CD28⁺ T cells were isolated by immunomagnetic bead depletion usingmAbs directed against B cells, natural killer cells, and macrophages aspreviously described (Gimmi, C. D., Freeman, G. J., Gribben, J. G.,Gray, G., Nadler, L. M. (1993) Proc. Natl. Acad. Sci USA 90, 6586-6590).B7-1, B7-2, and vector transfected COS cells were harvested 72 hoursafter transfection, incubated with 25 μg/ml of mitomycin-C for 1 hour,and then extensively washed. 10⁵ CD28⁺ T cells were incubated with 1ng/ml of phorbol myristic acetate (PMA) and 2×10⁴ COS transfectants.Blocking agents (10 μg/ml) are indicated on the left side of FIG. 12 andinclude: 1) no monoclonal antibody (no blocking agents), 2) mAb 133(anti-B7-1 mAb), 3) mAb BB1 (anti-B7-1 and anti-B7-3 mAb), 4) mAb B5(control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6) CTLA-Ig, and 7)control Ig. Panel a of FIG. 12 shows proliferation measured by ³H-thymidine (1 μCi) incorporation for the last 12 hours of a 72 hourincubation. FIG. 12, panels A-G, shows IL-2 production as measured byELISA (Biosource, Calif.) using supernatants harvested 24 hours afterthe initiation of culture.

To distinguish B7-2 from B7-1 and B7-3, mAbs directed against B7-1 andB7-3 were used to inhibit proliferation and IL-2 production ofsubmitogenically activated human CD28⁺ T cells. Both B7-1 and B7-2 COStransfectants costimulated T cell proliferation and IL-2 production(FIG. 12). MAbs 133 (Freedman, A. S. et al. (1987) supra) (anti-B7-1)and BB1 (Boussiotis, V. A., et al., (in review) Proc. Natl. Acad. Sci.USA; Yokochi, T., Holly, R. D., Clark, E. A. (1982) J. Immunol. 128,823-827) (anti-B7-1 and anti-B7-3) completely inhibited proliferationand IL-2 secretion induced by B7-1 but had no effect upon costimulationby B7-2 transfected COS cells. Isotype matched control B5 mAb had noeffect. To determine whether B7-2 signals via the CD28/CTLA4 pathway,anti-CD28 Fab and CTLA4-Ig fusion protein were tested to determinewhether they inhibited B7-2 costimulation. Both anti-CD28 Fab andCTLA4-Ig inhibited proliferation and IL-2 production induced by eitherB7-1 or B7-2 COS transfectants whereas control Ig fusion protein had noeffect (FIG. 12). While CTLA4-Ig inhibited B7-2 costimulation ofproliferation by only 90%, in other experiments inhibition was morepronounced (98-100%). None of the blocking agents inhibited T cellproliferation or IL-2 production induced by the combination of PMA andphytohemagglutinin.

Like B7-1, B7-2 is a counter-receptor for the CD28 and CTLA4 T cellsurface molecules. Both proteins are similar in that they are: 1)expressed on the surface of APCs; 2) structurally related to the Igsupergene family with an IgV and IgC domain which share 26% amino acididentity, and 3) capable of costimulating T cells to produce IL-2 andproliferate. However, B7-1 and B7-2 differ in several fundamental ways.First, B7-2 mRNA is constitutively expressed in unstimulated B cells,whereas B7-1 mRNA does not appear until 4 hours and cell surface proteinis not detected until 24 hours (Freedman, A. S., et al. (1987) supra;Freeman, G. J., et al. (1989) supra). Unstimulated human B cells do notexpress CTLA4 counter-receptors on the cell surface and do notcostimulate T cell proliferation (Boussiotis, V. A., et al. supra).Therefore, expression of B7-2 mRNA in unstimulated B cells would allowrapid expression of B7-2 protein on the cell surface followingactivation, presumably from stored mRNA or protein. Costimulation byB7-2 transfectants is partially sensitive to paraformaldehyde fixation,whereas B7-2 costimulation is resistant (Gimmi, C. D., et al. (1991)Proc. Natl. Acad. Sci. USA 88, 6575-6579). Second, expression of B7-1and B7-2 in cell lines and human B cell neoplasms substantially differs.Third, B7-2 protein contains a longer cytoplasmic domain than B7-1 andthis could play a role in signaling B-cell differentiation. Thesephenotypic and functional differences suggest that these homologousmolecules may have biologically distinct functions.

EXAMPLE 6 Cloning and Sequencing of the Murine B7-2 Antigen

A. Construction of cDNA Library

A cDNA library was constructed in the pCDM8 vector (Seed, Nature,329:840 (1987)) using poly (A)⁺ RNA from dibutryl cyclic AMP (cAMP)activated M12 cells (a murine B cell tumor line) as described (Aruffo etal, Proc. Natl. Acad. Sci. USA, 84:3365 (1987)).

M12 cells were cultured at 1×10⁶ cells/ml in complete culture media,{RPMI 1640 with 10% heat inactivated fetal calf serum (FCS), 2 mMglutamine, 1 mM sodium pyruvate, penicillin (100 units/ml), streptomycinsulfate (100 μg/ml) and gentamycin sulfate (5 μg/ml)}, in tissue cultureflasks and were activated by 300 μg/ml dibutryl cAMP (Nabavi, N., et al.(1992) Nature 360, 266-268). Activated M12 cells were harvested after 0,6, 12, 18, 24 and 30 hours.

RNA was prepared by homogenizing activated M12 cells in a solution of 4Mguanidine thiocyanate, 0.5% sarkosyl, 25 mM EDTA, pH 7.5, 0.13% Sigmaanti-foam A, and 0.7% mercaptoethanol. RNA was purified from thehomogenate by centrifugation for 24 hour at 32,000 rpm through asolution of 5.7M CsCl, 10 mM EDTA, 25 mM Na acetate, pH 7. The pellet ofRNA was dissolved in 5% sarkosyl, 1 mM EDTA, 10 mM Tris, pH 7.5 andextracted with two volumes of 50% phenol, 49% chloroform, 1% isoamylalcohol. RNA was ethanol precipitated twice. Poly (A)⁺ RNA used in cDNAlibrary construction was purified by two cycles of oligo (dT)-celluloseselection

Complementary DNA was synthesized from 5.5 μg of dibutryl cAMP activatedmurine M12 cell poly(A)⁺ RNA in a reaction containing 50 mM Tris, pH8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 500 μM dATP, dCTP,dGTP, dTTP, 50 μg/ml oligo(dT)₁₂₋₁₈, 180 units/ml RNasin, and 10,000units/ml Moloney-MLV reverse transcriptase in a total volume of 55 μl at37° C. for 1 hr. Following reverse transcription, the cDNA was convertedto double-stranded DNA by adjusting the solution to 25 mM Tris, pH 8.3,100 mM KCl, 5 mM MgCl₂, 250 μM each dATP, dCTP, dGTP, dTTP, 5 mMdithiothreitol, 250 units/ml DNA polymerase I, 8.5 units/ml ribonucleaseH and incubating at 16° C. for 2 hr. EDTA was added to 18 mM and thesolution was extracted with an equal volume of 50% phenol, 49%chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes ofethanol in the presence of 2.5M ammonium acetate and with 4 microgramsof linear polyacrylamide as carrier. Following reverse transcription,the reverse transcriptase was inactivated by heating at 70° C. for 10min. The cDNA was converted to double-stranded DNA by adding 320 μl H₂ Oand 80 μl of a solution of 0.1M Tris, pH 7.5, 25 mM MgCl₂, 0.5M KCl, 250μg/ml bovine serum albumin, and 50 mM dithiothreitol, and adjusting thesolution to 200 μM each dATP, dCTP, dGTP, dTTP, 50 units/ml DNApolymerase I, 8 units/ml ribonuclease H and incubating at 16° C. for 2hours. EDTA was added to 18 mM and the solution was extracted with anequal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA wasprecipitated with two volumes of ethanol in the presence of 2.5Mammonium acetate and with 4 micrograms of linear polyacrylamide ascarrier.

2 μg of non-selfcomplementary BstXI adaptors were added to the DNA asfollows: The double-stranded cDNA from 5.5 μg of poly(A)⁺ RNA wasincubated with 3.6 μg of a kinased oligonucleotide of the sequenceCTTTAGAGCACA (SEQ ID NO:15) and 2.4 μg of a kinased oligonucleotide ofthe sequence CTCTAAAG (SEQ ID NO:16) in a solution containing 6 mM Tris,pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovine serum albumin, 7 mMmercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1 mM spermidine, and600 units T4 DNA ligase in a total volume of 0.45 ml at 15° for 16hours. EDTA was added to 34 mM and the solution was extracted with anequal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA wasprecipitated with two volumes of ethanol in the presence of 2.5Mammonium acetate.

DNA larger than 600 bp was selected as follows: The adaptored DNA wasredissolved in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCl, 0.1% sarkosyland chromatographed on a Sepharose CL-4B column in the same buffer. DNAin the void volume of the column (containing DNA greater than 600 bp)was pooled and ethanol precipitated.

The pCDM8 vector was prepared for cDNA cloning by digestion with BstXIand purification on an agarose gel. Adaptored DNA from 5.5 μg ofpoly(A)⁺ RNA was ligated to 2.25 μg of BstXI cut pCDM8 in a solutioncontaining 6 mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovineserum albumin, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1mM spermidine, and 600 units T4 DNA ligase in a total volume of 1.5 mlat 15° for 24 hr. The ligation reaction mixture was transformed intocompetent E.coli MC1061/P3 and a total of 200×10⁶ independent cDNAclones were obtained.

Plasmid DNA was prepared from a 500 ml culture of the originaltransformation of the cDNA library. Plasmid DNA was purified by thealkaline lysis procedure followed by twice banding in CsCl equilibriumgradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1987)).

B. Cloning Procedure

In the first round of screening, thirty 100 mm dishes of 50% confluentCOS cells were transfected with 0.05 μg/ml activated M12 murine B celllibrary DNA using the DEAE-Dextran method (Seed et al, Proc. Natl. Acad.Sci. USA, 84:3365 (1987)). The cells were trypsinized and re-platedafter 24 hours. After 47 hours, the cells were detached by incubation inPBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37° C. for 30 min. Thedetached cells were treated with 10 μg/ml/human CTLA4Ig and murineCD28Ig for 45 minutes at 4° C. Cells were washed and distributed intopanning dishes coated with affinity-purified Goat anti-human IgGantibody and allowed to attach at room temperature. After 3 hours, theplates were gently washed twice with PBS/0.5 mM EDTA, pH 7.4/0.02% Naazide, 5% FCS and once with 0.15M NaCl, 0.01 M Hepes, pH 7.4, 5% FCS.Episomal DNA was recovered from the panned cells and transformed into E.coli DH10B/P3. The plasmid DNA was reintroduced into COS cells viaspheroplast fusion as described (Seed et al, Proc. Natl. Acad. Sci. USA,84:3365 (1987)) and the cycle of expression and panning was repeatedtwice. In the second and third rounds of selection, after 47 hours, thedetached COS cells were first incubated with α-murine B7-1 mAb (16-10A1,10 μg/ml), and COS cells expressing B7-1 were removed by α-mouse IgG andIgM coated magnetic beads. COS cells were then treated with 10 μg/ml ofhuman CTLA4Ig and murine CD28Ig and murine B7-2 expressing COS cellswere selected by panning on dishes coated with goat anti-human IgGantibody. After the third round, plasmid DNA was prepared fromindividual colonies and transfected into COS cells by the DEAE-Dextranmethod. Expression of B7-2 on transfected COS cells was analyzed byindirect immunofluorescence with CTLA4Ig.

After the final round of selection, plasmid DNA was prepared fromindividual colonies. A total of 6 of 8 candidate clones contained a cDNAinsert of approximately 1.2 kb. Plasmid DNA from these eight clones wastransfected into COS cells. All six clones with the 1.2 Kb cDNA insertwere strongly positive for B7-2 expression by indirectimmunofluorescence using CTLA4Ig and flow cytometric analysis.

C. Sequencing

The B7-2 cDNA insert in clone4 was sequenced in the pCDM8 expressionvector employing the following strategy. Initial sequencing wasperformed using sequencing primers T7, CDM8R (Invitrogen) homologous topCDM8 vector sequences adjacent to the cloned B7-2 cDNA (see Table II).Sequencing was performed using dye terminator chemistry and an ABIautomated DNA sequencer. (ABI, Foster City, Calif.). DNA sequenceobtained using these primers was used to design additional sequencingprimers (see Table II). This cycle of sequencing and selection ofadditional primers was continued until the murine B7-2 cDNA wascompletely sequenced on both strands.

                  TABLE II                                                        ______________________________________                                        T7 (F) (SEQ ID NO:3)                                                                              5'd[TAATACGACTCACTATAGGG]3'                               CDM8 (R)                                                                             (SEQ ID NO:4)                                                                               5'd[TAAGGTTCCTTCACAAAG]3'                                MBX4-1F                                                                              (SEQ ID NO:24)                                                                             5'd[ACATAAGCCTGAGTGAGCTGG]3'                              MBX4-2R                                                                              (SEQ ID NO:25)                                                                             5'd[ATGATGAGCAGCATCACAAGG]3'                              MBX4-14                                                                              (SEQ ID NO:26)                                                                             5'd[TGGTCGAGTGAGTCCGAATAC]3'                              MBX4-2F                                                                              (SEQ ID NO:27)                                                                             5'd[GACGAGTAGTAACATACAGTG]3'                              ______________________________________                                    

A murine B7-2 clone (mB7-2, clone 4) was obtained containing an insertof 1,163 base pairs with a single long open reading frame of 927nucleotides and approximately 126 nucleotides of 3' noncoding sequences(FIG. 14, SEQ ID NO:22). The predicted amino acid sequence encoded bythe open reading frame of the protein is shown below the nucleotidesequence in FIG. 14. The encoded murine B7-2 protein, is predicted to be309 amino acid residues in length (SEQ ID NO:23). This protein sequenceexhibits many features common to other type I Ig superfamily membraneproteins. Protein translation is predicted to begin at the methioninecodon (ATG, nucleotides 111 to 113) based on the DNA homology in thisregion with the consensus eucaryotic translation initiation site (seeKozak, M. (1987) Nucl. Acids Res. 15:8125-8148). The amino terminus ofthe murine B7-2 protein (amino acids 1 to 23) has the characteristics ofa secretory signal peptide with a predicted cleavage between the alanineat position 23 and the valine at position 24 (von Heijne (1987) Nucl.Acids Res. 14:4683). Processing at this site would result in a murineB7-2 membrane bound protein of 286 amino acids having an unmodifiedmolecular weight of approximately 32 kDa. This protein would consist ofan approximate extracellular Ig superfamily V and C like domains of fromabout amino acid residue 24 to 246, a hydrophobic transmembrane domainof from about amino acid residue 247 to 265, and a long cytoplasmicdomain of from about amino acid residue 266 to 309. The homologies tothe Ig superfamily are due to the two contiguous Ig-like domains in theextracellular region bound by the cysteines at positions 40 to 110 and157 to 216. The extracellular domain also contains nine potentialN-linked glycosylation sites and, like murine B7-1, is probablyglycosylated. Glycosylation of the murine B7-2 protein may increase themolecular weight to about 50-70 kDa. The cytoplasmic domain of murineB7-2 contains a common region which has a cysteine followed bypositively charged amino acids which presumably functions as signalingor regulatory domain within an APC. Comparison of both the nucleotideand amino acid sequences of murine B7-2 with the GenBank and EMBLdatabases yielded significant homology (about 26% amino acid sequenceidentity) with human and murine B7-1. Murine B7-2 exhibits about 50%identity and 67% similarity with its human homologue, hB7-2. E. coli(DH106/p3) transfected with a vector (plasmid pmB×4) containing a cDNAinsert encoding murine B7-2 (clone 4) was deposited with the AmericanType Culture Collection (ATCC) on Aug. 18, 1993 as Accession No. 69388.

D. Costimulation

CD4⁺ murine T cells were purified by first depleting red blood cells bytreatment with Tris-NH₄ Cl. T cells were enriched by passage over anylon wool column. CD4⁺ T cells were purified by two-fold treatment witha mixture of anti-MHC class II and anti-CD28 mAbs and rabbit complement.Murine B7-1 (obtained from Dr. Gordon Freeman, Dana-Farber CancerInstitute, Boston, Mass.; see also, Freeman, G. J. et al (1991) J. Exp.Med. 174, 625-631) murine B7-2, and vector transfected COS cells wereharvested 72 hours after trnasfection, incubated with 25 μg/mlmitomycin-C for one hour, and then extensively washed. 10⁵ murine CD4⁺ Tcells were incubated with 1 ng/ml of phorbol myristic acid (PMA) and2×10⁴ COS transfectants (Table III). T cell proliferation was measuredby ³ H-thymidine (1 μCi) incorporated for the last 12 hours of a 72 hourincubation.

                  TABLE III                                                       ______________________________________                                                          3H-Thymidine                                                                  Incorporation (cpm)                                         ______________________________________                                        CD4.sup.+  T cells     175                                                    CD4.sup.+  T cells + 1 ng/ml PMA                                                                     49                                                     CD4.sup.+  T cells + COS-vector                                                                      1750                                                   CD4.sup.+  T cells + COS-B7-1                                                                        4400                                                   CD4.sup.+  T cells + COS-B7-2                                                                        2236                                                   CD4.sup.+  T cells + 1 ng/ml PMA + COS-vector                                                        2354                                                   CD4.sup.+  T cells + 1 ng/ml PMA + COS-B7-1                                                          67935                                                  CD4.sup.+  T cells + 1 ng/ml PMA + COS-B7-2                                                          43847                                                  ______________________________________                                    

EXAMPLE 7 Construction and Characterization of Human B7-2 ImmunoglobulinFusion Proteins

A. Preparation Of Human B7-2Ig Fusion Proteins

The extracellular portion of human B7-2 was prepared as a fusion proteincoupled to an immunoglobulin constant region. The immunoglobulinconstant region may contain genetic modifications including those whichreduce or eliminate effector activity inherent in the immunoglobulinstructure. Briefly, DNA encoding the extracellular portion of hB7-2 wasjoined to DNA encoding the hinge, CH2 and CH3 regions of human IgCγ1 orIgCγ4 modified by directed mutagenesis. This was accomplished asdescribed in the following subsections.

B. Preparation of Gene Fusions

DNA fragments corresponding to the DNA sequences of interest wereprepared by polymerase chain reaction (PCR) using primer pairs describedbelow. In general, PCR reactions were prepared in 100 μl final volumecomposed of Taq, polymerase buffer (Gene Amp PCR Kit,Perkin-Elmer/Cetus, Norwalk, Conn.) containing primers (1 μM each),dNTPs (200 μM each) 1 ng of template DNA, and Taq, polymerase (Saiki, R.K., et al. (1988) Science 239:487-491). PCR DNA amplifications were runon a thermocycler (Ericomp, San Diego, Calif.) for 25 to 30 cycles eachcomposed of a denaturation step (1 minute at 94° C.), a renaturationstep (30 seconds at 54° C.), and a chain elongation step (1 minute at72° C.). The structure of each hB7-2 Ig genetic fusion consisted of asignal sequence to facilitate secretion coupled to the extracellulardomain of B7-2 and the hinge, CH2 and CH3 domains of human IgCγ1 orIgCγ4. The IgC gamma 1 and IgC gamma 4 sequences contained nucleotidechanges within the hinge region to replace cysteine residues availablefor disulfide bond formation with serine residues and may containnucleotide changes to replace amino acids within the CH2 domain thoughtto be required for IgC binding to Fc receptors and complementactivation.

Sequence analysis confirmed structures of both mγ₄ and γ₁ clones, andeach construct was used to transfect 293 cells to test transientexpression. hIgG ELISA measured/confirmed transient expression levelsapproximately equal to 100 ng protein/ml cell supernatant for bothconstructs. NSO cell lines were transfected for permanent expression thethe fusion proteins.

C. Genetic Construction of hB7-2Ig Fusion Proteins

(1). Preparation of Signal Sequence

PCR amplification was used to generate an immunoglobulin signal sequencesuitable for secretion of the B7-2Ig fusion protein from mammaliancells. The Ig signal sequence was prepared from a plasmid containing themurine IgG heavy chain gene (Orlandi, R. et al. (1989) Proc. Natl. Acad.Sci. USA. 86:38333837) using the oligonucleotide5'-GGCACTAGGTCTCCAGCTTGAGATCACAGTTCTCTCTAC-3' (#01) (SEQ ID NO:32) asthe forward primer and the oligonucleotide5'-GCTTGAATCTTCAGAGGAGCGGAGTGGACACCTGTGG-3' (#02) (SEQ ID NO:33) as thereverse PCR primer. The forward PCR primer (SEQ ID NO:32) containsrecognition sequences for restriction enzymes BsaI and is homologous tosequences 5' to the initiating methionine of the Ig signal sequence. Thereverse PCR primer (SEQ ID NO:33) is composed of sequences derived fromthe 5' end of the extracellular domain of hB7-2 and the 3' end of the Igsignal sequence. PCR amplification of the murine Ig signal template DNAusing these primers resulted in a 224 bp product which is composed ofBsaI restriction sites followed by the sequence of the Ig signal regionfused to the first 20 nucleotides of the coding sequence of theextracellular domain of hB7-2. The junction between the signal sequenceand hB7-2 is such that protein translation beginning at the signalsequence will continue into and through hB7-2 in the correct readingframe.

(2). Preparation of the hB7-2 Gene Segment

The extracellular domain of the hB7.2 gene was prepared by PCRamplification of plasmid containing the hB7-2 cDNA inserted intoexpression vector pCDNAI (Freeman et al., Science 262:909-11 (1994)):

The extracellular domain of hB7-2 was prepared by PCR amplificationusing oligonucleotide 5'-GCTCCTCTGAAGATTCAAGC-3' (#03) (SEQ ID NO:34) asthe forward primer and oligonucleotide5'-GGCACTATGATCAGGGGGAGGCTGAGGTCC-3' (#04) (SEQ ID NO:35) as the reverseprimer. The forward PCR primer contained sequences corresponding to thefirst 20 nucleotides of the B7-2 extracellular domain and the reversePCR primer contained sequences corresponding to the last 22 nucleotidesof the B7-2 extracellular domain followed by a Bcl I restriction siteand 7 noncoding nucleotides. PCR amplification with primer #03 (SEQ IDNO:34) and #04 (SEQ ID NO:35) yields a 673 bp product corresponding tothe extracellular IgV and IgC like domains of hB7-2 followed by a uniqueBcl I restriction site.

The signal sequence was attached to the extracellular portion of hB7-2by PCR as follows. DNA-PCR products obtained above corresponding to thesignal sequence and the hB7-2 extracellular domain were mixed inequimolar amounts, denatured by heating to 100° C., held at 54° C. for30° C. to allow the complementary ends to anneal and the strands werefilled in using dNTPs and Tag polymerase. PCR primers #01 (SEQ ID NO:32)and #04 (SEQ ID NO:35) were added and the entire fragment produced byPCR amplification to yield a 880 fragment composed of a BsaI restrictionsite followed by the signal sequence fused to the extracellular domainof hB7-2, followed by a Bcl I restriction site.

(3). Cloning and Modification of Immunozlobulin Fusion Domain

Plasmid pSP721gG1 was prepared by cloning the 2000 bp segment of humanIgG1 heavy chain genomic DNA (Ellison, J. W., et al. (1982) Nucl. Acids.Res. 10:4071-4079) into the multiple cloning site of cloning vectorpSP72 (Promega, Madison, Wis.). Plasmid pSP721gG1 contained genomic DNAencoding the CH1, hinge, CH2 and CH3 domains of the heavy chain humanIgCγ1 gene. PCR primers designed to amplify the hinge-CH2-CH3 portion ofthe heavy chain along with the intervening DNA were prepared as follows.The forward PCR primer 5'-GCATTTTAAGCTTTTTCCTGATCAGGAGCCCAAATCTTCTGACAAAACTCACACATCTCCACCGTCTCCAGGTAAGCC-3' (SEQ ID NO:36) containedHindIII and Bcl I restriction sites and was homologous to the hingedomain sequence except for five nucleotide substitutions which wouldchange the three cysteine residues to serines. The reverse PCR primer5'TAATACGACTCACTATAGGG-3' (SEQ ID NO:37) was identical to thecommercially available T7 primer (Promega, Madison, Wis.). Amplificationwith these primers yielded a 1050 bp fragment bounded on the 5' end byHindIII and Bcl I restriction sites and on the 3'end by BamHl , Smal,Kpnl, Sacl, EcoR1, Clal, EcoR5 and BgIII restriction sites. Thisfragment contained the IgC hinge domain in which the three cysteinecodons had been replaced by serine codons followed by an intron, the CH2domain, an intron, the CH3 domain and additional 3' sequences. After PCRamplification, the DNA fragment was digested with HindIII and EcoR1 andcloned into expression vector pNRDSH digested with the same restrictionenzymes. This created plasmid pNRDSH/IgG1.

A similar PCR based strategy was used to clone the hinge-CH2-CH3 domainsof human IgCgamma4 constant regions. A plasmid, p428D (Medical ResearchCouncil, London, England) containing the complete IgCgamma4 heavy chaingenomic sequence (Ellison, J. Buxbaum, J. and Hood, L. E. (1981) DNA 1:11-18) was used as a template for PCR amplification usingoligonucleotide 5'GAGCATTTTCCTGATCAGGAGTCCAAATATGGTCCCCCATCCCATCATCCCCAGGTAAGCCAACCC-3' (SEQ ID NO:38) as theforward PCR primer and oligonucleotide5'GCAGAGGAATCGAGCTCGGTACCCGGGGATCCCCAGTGTGGGGACAGTGGGA CCGCTCTGCCTCCC-3'(SEQ ID NO:39) as the reverse PCR primer. The forward PCR primer (SEQ IDNO:38) contains a Bcl I restriction site followed by the coding sequencefor the hinge domain of IgCgamma4. Nucleotide substitutions have beenmade in the hinge region to replace the cysteines residues with serines.The reverse PCR primer (SEQ ID NO:39) contains a PspAI restriction site(5'CCCGGG-3'). PCR amplification with these primers results in a 1179 bpDNA fragment. The PCR product was digested with Bcll and PspAI andligated to pNRDSH/IgG1 digested with the same restriction enzymes toyield plasmid pNRDSH/IgG4. In this reaction, the IgCγ4 domain replacedthe IgCγ1 domain present in pNRDSH/IgG1.

Modification of the CH2 domain in IgC to replace amino acids thought tobe involved in binding to Fc receptor was accomplished as follows.Plasmid pNRDSH/IgG1 served as template for modifications of the IgCγ1CH2 domain and plasmid pNRDSH/IgG4 served as template for modificationsof the IgCγ4 CH2 domain. Plasmid pNRDSH/IgG1 was PCR amplified using aforward PCR primer (SEQ ID NO:36) and oligonucleotide 5'-GGGTTTTGGGGGGAAGAGGAAGACTGACGGTGCCCCC TCGGCTTCAGGTGCTGAGGAAG-3' (SEQ ID NO:40)as the reverse reverse primer. The forward PCR primer (SEQ ID NO:36) hasbeen previously described and the reverse primer (SEQ ID NO: ) washomologous to the amino terminal portion of the CH2 domain of IgG1except for five nucleotide substitutions designed to change amino acids234, 235, and 237 (Canfield, S. M. and Morrison, S. L. (1991) J. Exp.Med. 173: 1483-1491.) from Leu to Ala, Leu to Glu, and Gly to Ala,respectively. Amplification with these PCR primers will yield a 239 bpDNA fragment consisting of a modified hinge domain, an intron andmodified portion of the CH2 domain. Plasmid pNRDSH/IgG1 was also PCRamplified with the oligonucleotide5'-CATCTCTTCCTCAGCACCTGAAGCCGAGGGGGCACCGTCAGTCTTCCTCTTCCC CC-3' (SEQ IDNO:41) as the forward primer and oligonucleotide (SEQ ID NO:37) as thereverse PCR primer. The forward PCR primer (SEQ ID NO:41) iscomplementary to primer (SEQ ID NO:40) and contains the fivecomplementary nucleotide changes necessary for the CH2 amino acidreplacements. The reverse PCR primer (SEQ ID NO:37) has been previouslydescribed. Amplification with these primes yields a 875 bp fragmentconsisting of the modified portion of the CH2 domain, an intron, the CH3domain, and 3' additional sequences. The complete IgCγ1 segmentconsisting of modified hinge domain, modified CH2 domain and CH3 domainwas prepared by an additional PCR reaction. The purified products of thetwo PCR reactions above were mixed, denatured (95° C., 1 minute) andthen renatured (54° C., 30 seconds) to allow complementary ends of thetwo fragments to anneal. The strands were filled in using dNTP and Taqpolymerase and the entire fragment amplified using forward PCR primer(SEQ ID NO:36) and reverse PCR primer (SEQ ID NO:37). The resultingfragment of 1050 bp was purified, digested with HindIII and EcoR1 andligated to pNRDSH previously digested with the same restriction enzymesto yield plasmid pNRDSHIgG1 m.

Two amino acids at immunoglobulin positions 235 and 237 were changedfrom Leu to Glu and Gly to Ala, respectively, within the IgCγ4 CH2domain to eliminate Fc receptor binding. Plasmid pNRDSH/IgG4 was PCRamplified using the forward primer (SEQ ID NO:38) and theoligonucleotide5'-CGCACGTGACCTCAGGGGTCCGGGAGATCATGAGAGTGTCCTTGGGTTTTGGGGGGAACAGGAAGACTGATGGTGCCCCCTCGAACTCAGGTGCTGAGG-3' (SEQ ID NO:42) as thereverse primer. The forward primer has been previously described and thereverse primer was homologous to the amino terminal portion of the CH2domain, except for three nucleotide substitutions designed to replacethe amino acids described above. This primer also contained a Pmllrestriction site for subsequent cloning. Amplification with theseprimers yields a 265 bp fragment composed of the modified hinge region,and intron, and the modified 5' portion of the CH2 domain.

Plasmid pNRDSH/IgG4 was also PCR amplified with the oligonucleotide5'-CCTCAGCACCTGAGTTCGAGGGGGCACCATCAGTCTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG-3' (SEQ ID NO:43) asthe forward primer and oligonucleotide (SEQ ID NO:39) as the reverse PCRprimer. The forward PCR primer (SEQ ID NO:43) is complementary to primer(SEQ ID NO:42) and contains the three complementary nucleotide changesnecessary for the CH2 amino acid replacements. The reverse PCR primer(SEQ ID NO:39) has been previously described. Amplification with theseprimers yields a 1012 bp fragment consisting of the modified portion ofthe CH2 domain, an intron, the CH3 domain, and 3' additional sequences.The complete IgCγ4 segment consisting of modified hinge domain, modifiedCH2 domain and CH3 domain was prepared by an additional PCR reaction.The purified products of the two PCR reactions above were mixed,denatured (95° C., 1 minute) and then renatured (54° C., 30 seconds) toallow complementary ends of the two fragments to anneal. The strandswere filled in using dNTP and Taq polymerase and the entire fragmentamplified using forward PCR primer (SEQ ID NO:38) and reverse PCR primer(SEQ ID NO:39). The resulting fragment of 1179 bp was purified, digestedwith Bcll and PspAI and ligated to pNRDSH previously digested with thesame restriction enzymes to yield plasmid pNRDSH/IgG4m.

(4). Assembly of Final hB7-2Ig Genes

The PCR fragment corresponding to the Ig signal-hB7-2 gene fusionprepared above was digested with Bsal and Bcl I restriction enzymes andligated to pNRDSH/IgG1, pNRDSH/IgG1m, pNRDSH/IgG4, and pNRDSH/IgG4mpreviously digested with Hind III and BclI. The ligated plasmids weretransformed into E. coli JM109 using CaCl2 competent cells andtransformants were selected on L-agar containing ampicillin (50 μg/ml;Molecular Cloning: A Laboratory Manual (1982) Eds. Maniatis, T.,Fritsch, E. E., and Sambrook, J. Cold Spring Harbor Laboratory).Plasmids isolated from the transformed E. coli were analyzed byrestriction enzyme digestion. Plasmids with the expected restrictionplasmid were sequenced to verify all portions of the signal-hB7-2-IgGgene fusion segments.

D. Expression Cloning of hB7-2V-IgG1 and hB7-2C IgG1

The variable and constant domains of human B7-2 were separately clonedinto pNRDSH/IgG1. These clonings were accomplished using PCR. Theportions of hB7-2 corresponding to the variable and constant regionswere determined from intron/exon mapping and previously published genestructure analysis.

    Human B7-2 Variable Domain                                                          5'GCTCCTCTGAAGATT....................GAACTGTCAGTGCTT3'                                                       (SEQ ID NO:44)                                    A  P  L  K  I                  (SEQ ID NO:45)  L                     Human B7-2 Constant Domain                                                          5'GCTAACTTCAGTCAA....................CCTTTCTCTATAGAG3'                                                       (SEQ ID NO:46)                                    A  N  F  S  Q                  (SEQ ID NO:47)  E                 

(1). Assembly of hB7-2VIg

The hB7-2V domain Ig sequence was assembled using a PCR strategy similarto that shown above. The signal sequence was derived from the onco Mgene by PCR amplification of a plasmid containing the onco M gene usingoligonucleotide 5'-GCAACCGGAAGCTTGCCACCATGGGGGTACTGCTCACACAGAGGACG-3'(#05) (SEQ ID NO:48) as the forward PCR primer and5'-AGTCTCATTGAAATAAGCTTGAATCTTCAGAGGAGCCATGCTGGCCATGCTTGGA AACAGGAG-3'(#06) (SEQ ID NO:49) as the reverse primer. The forward PCR primer (#05)(SEQ ID NO:48) contains a Hind III restriction site and the aminoterminal portion of the onco M signal sequence. The reverse PCR (#06)(SEQ ID NO:49) contains the sequence corresponding to the 3' portion ofthe onco M signal sequence fused to the 5' end of the hB7-2 IgV likedomain.

The hB7-2 IgV like domain was obtained by PCR amplification of the hB7-2cDNA using oligonucleotide 5'-CTCCTGTTTCCAAGCATGGCCAGCATGGCTCCTCTGAAGATTCAGGCTTATTTCAATGAGAC-3' (#07) (SEQ ID NO:50) as the forward andoligonucleotide5'-TGTGTGTGGAATTCTCATTACTGATCAAGCACTGACAGTTCAGAATTCATC-3' (#08) (SEQ IDNO:51) as the reverse PCR primer. PCR amplification with these primersyields the hB7-2 IgV domain with a portion of the 3' end of the onco Msignal sequence on the 5' end and a Bcl I restriction site on the 3'end. The signal and IgV domain were linked together in a PCR reaction inwhich equimolar amounts of the onco M signal and IgV domain DNAfragments were mixed, denatured, annealed, and the strands filled in.Subsequent PCR amplification using forward primer #05 (SEQ ID NO:48) andreverse primer #08 (SEQ ID NO:51) yielded a DNA fragment containing aHind III restriction site, followed by the onco M signal fused to theB7-2 IgV domain followed by a Bcl I restriction site. This PCR fragmentwas digested with Hind II and Bcl I and cloned into expression vectorpNRDSH/IgG1 digested with the same restriction enzymes to yieldpNRDSH/B7-2CIg.

(2). Assembly of hB7-2CIg

The expression plasmid for hB7-2IgC domain was prepared as describedabove for the IgV domain except for using PCR primers specific for theIgC domain. The onco M signal sequence was prepared usingoligonucleotide #05 SEQ ID NO:48 as the forward PCR primer andoligonucleotide5'-AGAAATTGGTACTATTTCAGGTTGACTGAAGTTAGCCATGCTGGCCATGCTTGGA AACAGGAG-3'(#09) (SEQ ID NO:52) as the reverse PCR primer. The hB7-2 IgC domain wasprepared using oligonucleotide5'-CTCCTGTTTCCAAGCATGGCCAGCATGGCTAACTTCAGTC AACCTGAAATAGTACCAATTTC-3'(#11) (SEQ ID NO:53) as the reverse PCR primer. The two PCR productswere mixed and amplified with primers #05 (SEQ ID NO:48) and #11 (SEQ IDNO:53) to assemble the onco M signal sequence with the hB7-2IgC domain.The PCR product was subsequently digested with Hind III and BclI andligated to pNRDSH/IgG1 digested with similar restriction enzymes toyield the final expression plasmid pNRDSH/hB7-2CIgG1.

E. Competitive Binding Assays with Human B7-2Ig Fusion Proteins

To determine the affinity of binding of different forms of soluble B7-1and B7-2 proteins to CTLA4, competitive binding assays were performedwith these proteins. The soluble B7-2VIg, B7-2CIg, B7-2Ig, and B7-1Igfusion proteins used in these assays were expressed and purified asfollows.

The preparation of expression vectors encoding human B7-2VIg, B7-2CIg,and B7-2Ig fusion proteins is described above. The expression vectorencoding the B7-1Ig fusion protein, containing an OncoM leader sequencelinked the extracellular domain of B7-1 was prepared similarly, usingthe PCR primers OncoMB71F (5'CTCAAGCTTGCCACCATGGGGGTACTGCTCACACAGAGGACGCTGCTCAGTCTGGTCCTTGCACTCCTGTTTCCGAGCATGGCGAGCATGGGTCTTTC TCACTTC3'; SEQID NO:54) and B71/BclI (5'TGTGTGTGGAATTCTCATTACTGATCAGGAAAATGCTCTTGCTTG3'; SEQ ID NO:55). The plasmid pKShB7-1,containing the OncoM leader sequence linked to the human B7-1 cDNAsequence (the nucleotide sequence of the human B7-1 cDNA is disclosed inFreeman, G. J. et al., (1989) J. Immunol. 143:2714-2722) was used as atemplate in this PCR reaction.

The B7Ig fusion proteins were prepared by transfection of COS cells orChinese Hamster Ovary (CHO) cells and purification of the protein fromthe supernatant of the cultures.

Cell culture reagents were obtained from Gibco-BRL, Gaithersburg, Md.CHO cells were maintained in alpha MEM supplemented with 10% Fetalbovine serum (FBS) and glutamine. Penicillin, streptomycin, andfungizone were typically added. COS cells were maintained in DMEM with10% FBS and supplemented as described for CHO cells. All cells were keptat 5% CO2 at 37° C. in a humidified incubator.

All fusion constructs except the hB7-1Ig construct were expressedtransiently in COS cells. Typical transient transfections were doneusing 200 μgs/ml of DEAE-dextran, 100 μM chloroquine and 5 μgs of DNAper 10 cm dish in serum-free DMEM. The cells were treated until vacuoleswere noted and the cells appeared distressed (about 3 hours). Cells wereshocked with 10% DMSO/PBS for 2 minutes, then incubated with DMEM/10%FBS overnight. The following morning the media was changed to DMEM/serumfree and left until harvest at 72 hours post transfection. The hB7-1Igconstruct was transfected into CHO cells by calcium phosphatetransfection. The line was made stable using Geneticin (G418) resistanceselection, and expression was amplified using methotrexate and alpha MEMlacking nucleosides.

For all transiently transfected constructs (i.e., all constructs excepthB7-1Ig) media enriched for Ig fusion proteins produced by thetransiently transfected host cells was harvested 72 hourspost-transfection. The amount of Ig fusion proteins produced by the hostcells was measured by performing an anti-human IgG Elisa assay with thesupernatant of the cultures. For this assay, Maxisorp plates (Nunc,Denmark) were coated overnight with 20 μgs/ml of goat anti-human IgG(H+L) (Zymed, San Francisco, Calif.) in PBS. The plates were blocked for1 hour with PBS/0.1% BSA and then incubated for an additional hour withcell culture supernatants from the transfected cells. After 5 washeswith PBS/0.05% Tween, HRP-coupled goat anti-human IgG(H+L) (Zymed) wasadded as a 1:1000 dilution in PBS. After a 1 hour incubation the plateswere washed again, then enzymatically developed using an ABTS kit(Zymed) as described above.

Expression levels were approximately 3 μg/ml for the constructs. TheB7Ig fusion proteins were purified from the supernatant of transfectedhost cells by protein A purification as follows. Protein A SepharoseIPA300 (Repligen, Cambridge, Mass.) was washed in OBB (1.5 M glycine, 3M NaCl, pH 8.9), resuspended in DMEM, and added to cell culturesupernatant at 1 ml/liter of supernatant. The solution was left to mixgently overnight at 4° C. The Protein A sepharose was then pelleted, themajority of the supernatant was removed, and the remaining supernatantwas used to resuspend the Protein-A Sepharose prior to loading onto aPoly-prep column (BioRad, Hercules, Calif.). The column was washedextensively with OBB and the immunoglobulin fusion protein was eluted in0.1 M sodium citrate. Half volume column washes were collected into1/10th volume 1 M Tris (pH9.0). Protein containing fractions wereidentified by a standard colorimetric reaction (BioRad), pooled, anddialyzed overnight against PBS in 6000-8000 KD dialysis tubing. Thepurified proteins were of the expected size and high purity,representing >90% of total protein stained with Commassie Blue onacrylamide gels.

The ability of various B7 family-Ig fusion proteins to competitivelyinhibit the binding of biotinylated-CTLA4Ig to immobilized B7-2Ig wasdetermined. Competition binding assays were done as follows and analysedaccording to McPherson (McPherson, G. A. (1985) J. Pharmacol. Methods14:213-228). Soluble hCTLA4Ig was labelled with ¹²⁵ I to a specificactivity of approximately 2×10⁶ cpm/pmol. hB7-2-Ig fusion protein wascoated overnight onto microtiter plates at 10 mg/ml in 10 mM Tris-HCl,pH8.0, 50 ml/well. The wells were blocked with binding buffer (DMEMcontaining 10% heat-inactivated FBS, 0.1% BSA, and 50 mM BES, pH 6.8)for 2 h at room temperature. The labeled CTLA4-Ig (4 nM) was added toeach well in the presence or absence of unlabeled competing Ig fusionproteins, including full-length B7-2 (hB7-2Ig), full-length B7-1(hB7-1Ig), the variable region-like domain of B7-2 (hB7-2VIg) and theconstant region-like domain of B7-2 (hB7-2CIg) and allowed to bind for2.5 h at room temperature. The wells were washed once with ice-coldbinding buffer and then four times with ice-cold PBS. Boundradioactivity was recovered by treatment of the wells with 0.5 N NaOHfor 5 min and the solubilized material removed and counted in a gammacounter.

The results of these assays are shown in FIG. 15 in which both hB7-2Ig(10-20 nM) and hB7-2VIg (30-40 nM) competitively inhibit the binding ofCTLA4Ig to immobilized B7-2 protein. hB7-2CIg is unable to compete withsoluble CTLA4, indicating that the B7-2 binding region is in found inthe variable-region like domain.

F. Competitive Binding Assays for B7-1 and B7-2 Fusion Proteins

The ability of recombinant CTLA4Ig to bind to hB7-1 or hB7-2 wasassessed in a competitive binding ELISA assay as follows. Purifiedrecombinant CTLA4Ig (20 μg/ml in PBS) was bound to a Costar EIA/RIA 96well microtiter dish (Costar Corp, Cambridge Mass., USA) in 50 μLovernight at room temperature. The wells were washed three times with200 μL of PBS and the unbound sites blocked by the addition of 1% BSA inPBS (200 μl/well) for 1 hour at room temperature. The wells were washedas above. Biotinylated B7-1Ig or B7-2Ig (1 μg/ml serially diluted intwofold steps to 15.6 ng/mL; 50 μL) was added to each well and incubatedfor 2.5 hours at room temperature. The wells were washed as above. Thebound biotinylated B7-Ig was detected by the addition of 50 μl/well of a1:2000 dilution of streptavidin-HRP (Pierce Chemical Co., Rockford,Ill.) for 30 minutes at room temperature. The wells were washed as aboveand 50 μL of ABTS (Zymed, Calif.) added and the developing blue colormonitored at 405 nm after 30 min. The ability of unlabelled B7-1Ig orB7-2Ig to compete with biotinylated B7-1Ig or B7-2Ig, respectively, wasassessed by mixing varying amounts of the competing protein with aquantity of biotinylated B7-1Ig or B7-2Ig shown to be non-saturating(i.e., 70 ng/mL; 1.5 nM) and performing the binding assays as describedabove. A reduction in the signal (Abs 405 nm) expected for biotinylatedB7-1Ig or B7-2Ig indicated a competition for binding to immobilizedCTLA4Ig.

Considering the previous evidence that CTLA4 was the high affinityreceptor for B7-1, the avidity of binding of CTLA4 and CD28 to B7-1 andB7-2 was compared in assays as described above. In a first experiment,B7-1Ig or B7-2-Ig was labelled with biotin and bound to immobilizedCTLA4-Ig in the presence or absence of increasing concentrations ofunlabeled B7-1Ig or B7-2Ig. The experiment was repeated with ¹²⁵-I-labeled B7-1Ig or B7-2Ig. Representative results are shown in FIG. 16(Panel A: B7-1Ig; Panel B: B7-2Ig). Using this solid phase bindingassay, the avidity of B7-2 (2.7 nM) for CTLA4 was determined to beapproximately two-fold lower than that observed for B7-1 (4.6 nM). Theexperimentally determined IC₅₀ values are indicated in the upper rightcomer of the panels. The affinity of both B7-1 and B7-2 for CD28 waslower and was difficult to confidently determine.

G. Direct Binding Assays of modified forms of B7 family members toCTLA4Ig

Direct binding ELISA assays were performed to determine the level ofbinding of B7 family members as Ig fusions proteins to CTLA4. For theseassays, the immunoglobulin fusion proteins were attached to plates andthe amount of biotinylated CTLA4 binding to the plates was determined asdescribed below.

Nunc Maxisorp plates were coated overnight at room temperature with 50μl per well of a 20 μg/ml stock of the various B7Ig fusion proteins, orpurified human IgG (Zymed) in PBS as described above. Human CTLA4Ig(Repligen) was biotinylated using NHS-LC-biotin (Pierce, Rockford,Ill.). Varying amounts of biotinylated CTLA4Ig were added to the platesand incubated for 2 hours at room temperature. The plates were washedfive times with PBS and then a 1:1000 dilution of streptaviden-HRP(Zymed) was added and left for 30' minutes on the plates. After anotherseries of washes with PBS, the HRP reactivity was measured using an ABTSkit (Zymed) as described above.

The results, presented in FIG. 20, show that half-saturation occurred at500 pM for B7-1Ig, and at 5 nM and 8 nM for B7-2VIg and B7-2Ig,respectively. Thus, CTLA4Ig binds to a similar extent to the B7-2VIg andB7-2Ig fusion proteins. CTLA4 does not, however bind to B7-2CIg. Thus,the variable domain of B7-2 is sufficient for binding to CTLA4.

H. Binding of B7-2VIg, B7-2Ig. and B7-1Ig to CHO-CTLA4 cells

The examples presented in section F and G of Example 7 showed thatB7-2VIg and B7-2Ig bind soluble CTLA4Ig. The present example shows thatB7-2VIg and B7-2Ig also bind to CTLA4 expressed on a cell.

For this example, labeled B7-1Ig and B7-2Ig fusion proteins wereincubated with CHO cells transfected to express CTLA4 and binding wasmeasured by flow cytometry as follows.

B7-1 and B7-2 immunoglobulin fusion proteins, prepared as describedabove, were diluted to 20 μg/ml in PBS/1% BSA and incubated with 10⁶ CHOcells transfected to express CTLA4 on their surface (CHO/CTLA4 cells)for 30 minutes on ice. The cells were washed twice with cold PBS/BSA andincubated with a 1:50 dilution of goat anti-human IgG-FITC (Zymed) for30 minutes on ice. The cells were washed once with cold PBS/BSA, oncewith cold PBS and then resuspended in 250 μl cold PBS. The cells werethen fixed by adding 250 μl of a 2% paraformaldehyde solution in PBS andincubation for at least 1 hour and the fluorescence analyzed using aFACS (Becton Dickinson, San Jose, Calif.). Similarly treated CHO/CTLA4cells which recieved the secondary antibody alone served to measurebackground staining.

The results of the flow cytometric analysis are presented in FIG. 21.The results show that hB7-2Ig and hB7-2VIg fusion proteins bind to asimilar extent to CTLA4 positive cells (FIG. 21, panels C and D,repectively) and that the binding is stronger than binding of hB7-1Ig toCHO/CTLA4 cells (panel E).

I. B7-2VIg binds with stronger affinity to CD28 than B7-1Ig and B7-2Ig

The Example shown in the previous section showed that B7-2VIg and B7-2Igfusion proteins bind with similar affinity to cell membrane bound CTLA4.This example shows that the fusion proteins bind with differentaffinities to CD28 and in particular, that B7-2VIg binds with higheraffinity to CD28 than B7-2Ig.

B7-1 and B7-2 immunoglobulin fusion proteins diluted at 20 μg/ml inPBS/1% BSA were incubated with 10⁶ CHO cells transfected to express CD28on their surface (CHO/CD28 cells) for 30 minutes on ice. The cells werewashed twice with cold PBS/BSA and incubated with a 1:50 dilution ofgoat anti-human IgG-FITC (Zymed) for 30 minutes on ice. The cells werewashed once with cold PBS/BSA, once with cold PBS and then resuspendedin 250 μl cold PBS. The cells were then fixed by adding 250 μl of a 2%paraformaldehyde solution in PBS and incubation for at least 1 hour andthe fluorescence analyzed using a FACS (Becton Dickinson, San Jose,Calif.).

Representative results, as presented in FIG. 22, indicate that B7-2Igand B7-2VIg fusion proteins bind specifically to CHO-CD28 cells. Theresults further indicate that B7-2VIg protein binds to CD28 withstronger affinity than does B7-2Ig and B7-1Ig.

Thus, B7-2VIg fusion protein binds with higher affinity than B7-2Ig toCD28, whereas both fusion proteins bind with similar affinity to CTLA4.

J. B7-2VIg is more potent than B7-2Ig and B7-1Ig at costimulatingproliferation of CD28+ T cells

Since B7-2VIg binds with higher affinity to CD28 than B7-2Ig, it wasnext investigated whether B7-2VIg fusion protein is also more potent atstimulating T cell proliferation than B7-2Ig fusion protein.

CD28+ T cells were isolated from peripheral blood leukocytes (PBLs) asdescribed above. For measuring T cell proliferation, 1.2×10⁵ CD28+ Tcells were incubated in 200 μl of culture media in 96 well plates andstimulated with PMA at 1 ng/ml and either of the following costimulatorysignals: 6×10⁴ CHO/B7-1 or CHO/B7-2 cells (pretreated overnight withmitomycin C and then extensively washed), 30 or 100 μg/ml of B7-1Ig,B7-2Ig, or B7-2VIg. Alternatively, the fusion proteins can first beincubated with a 3 fold excess (w/w) of affinity purified goatanti-human IgGFc (Cappel) for 30 minutes prior to use. After 60 hours ofincubation, the T cells were pulsed overnight with ³ H-thymidine(Dupont/NEN) and harvested for counting, as described above.

The results of the proliferation assay are represented graphically inFIG. 23. The results indicate that CHO expressed B7-1 and B7-2 stronglyinduced T cell proliferation. Purified B7-1Ig and B7-2Ig also inducedproliferation, although not as potently as the CHO/B7-1 and CHO/B7-2cells. However, B7-2VIg induced proliferation to the same extent as theCHO/B7-1 and CHO/B7-2 cells. Thus, B7-2VIg is as potent as CHO/B7-1 andCHO/B7-2 cells in costimulating proliferation of T cells.

It another example, CD28+ T cells were activated with anti-CD3 coatedplates prepared as described above and costimulated with various amountsof B7-1Ig, B7-2Ig , and B7-2VIg. Proliferation of the CD28+ T cells wasmeasured after 60 hours and overnight pulsing of the cells with ³H-thymidine. The results, presented graphically in FIG. 24 indicate thatB7-2VIg fusion protein is also more potent than B7-2Ig and B7-1Ig atcostimulating CD28+ T cells when anti-CD3 is used as the primaryactivating agent. Moreover, the results indicate that B7-2VIg is morepotent than B7-2Ig and B7-1Ig at costimulating proliferation of CD28+ Tcells when low concentrations of the proteins are used. This is apparentwhen comparing the amount of thymidine incorporated in cells incubatedwith 1 μg/ml of costimulatory fusion protein. In addition, B7-2VIgcostimulates proliferation of the T cells at doses as low as 10 ng/ml(250 pM).

Thus, the higher binding affinity of B7-2VIg versus B7-2Ig fusionprotein for CD28 (Example 7, section I) correlates with a highercostimulatory activity of B7-2VIg versus B7-2Ig for proliferatin of theT cells.

K. B7-2VIg is more potent than B7-1Ig and B7-2Ig at costimulatingproduction of IL-2 by CD28+ T cells

It was next investigated whether B7-2VIg was also more potent thanB7-1Ig and B7-2Ig at costimulating activated T cells for the productionof IL-2.

In the second example described in the previous section (Section J ofExample 7), in which CD28+ T cells were activated with anti-CD3 andcostimulated with various amounts of B7-1Ig, B7-2Ig, and B7-2VIg, andcell proliferation measured, the level of IL-2 in the supernatant wasdetermined after 18 hours of stimulation using an ELISA kit (Endogen,Cambridge, Mass.). The results, presented in FIG. 24 show that more IL-2is produced by T cells costimulated with B7-2VIg than by T cellscostimulated with B7-1Ig or B7-2Ig. This was most apparent at lowconcentrations of costimulatory proteins (for example at 1 μg/ml of thefusion proteins).

Another example was performed to compare production of IL-2 from CD28+ Tcells costimulated with CHO/B7-2 cells or costimulated with B7-2VIgprotein. CD28+ T cells were incubated in anti-CD3 coated plates in thepresence of CHO/B7-2 cells or B7-2VIg protein for 1, 2, or 3 days, andthe amount of IL-2 was measured in the supernatant. The results,presented in FIG. 25, show that B7-2VIg is more potent at costimulatingT cells for the production of IL-2 than CHO/B7-2 cells.

In a further example, the amount of IL-2 produced by CD28+ T cellscostimulated with anti-CD28, B7-1Ig, B7-2Ig, or B7-2VIg was comparedafter 1, 2, or 5 days of costimulation. CD28+ T cells were activated andcostimulated with anti-CD28 antibody, or with B7-1Ig, B7-2Ig, or B7-2VIgfusion protein. The amount of IL-2 in the supernatant was measured after1, 2, and 5 days of costimulation. FIG. 26, representing graphically theamount of IL-2 produced by the T cells, indicate that B7-2IVIg is morepotent than B7-2Ig and B7-1Ig at stimulating production of IL-2 by CD28+T cells and further, that after 5 days of culture, only T cellscostimulated with B7-2Ig or B7-2VIg fusion proteins were producing IL-2.Moreover, T cells costimulated with B7-2VIg produce more IL-2 than Tcells costimulated with B7-2Ig after 5 days of culture.

Thus, B7-2VIg costimulate T cells to produce high levels of IL-2, evenafter at least 5 days of culture.

L. B7-2VIg is a potent costimulator of activated CD4+ T cells for theproduction of cytokines.

In this example, the amount of IL-2, IL-4, IFN-γ, and GM-CSF produced byT cells costimulated with B7-2VIg, B7-2Ig, or B7-1Ig was compared.

CD4+CD28+ T cells were cultured at 2×10⁶ cells/ml in T75 flasks coatedwith anti-CD3 antibody alone, or with anti-CD28 mAb 9.3, or one of thefusion proteins B7-1Ig, B7-2Ig, or B7-2VIg. After 18 hours of culture,the amount of IL-2, IL-4, interferon-γ (IFN-γ), and granulocytemacrophage-colony stimulating factor (GM-CSF) was measured by ELISAusing commercially available kits (IL-2 (BioSource, Camarillo, Calif.),IL-4 (Endogen, Cambridge, Mass.), IFN-γ (Bio-Source, Camarillo, Calif.),and GM-CSF (R&D Systems, Minneapolis, Minn.)). The amount of IL-2 andIL-4 was also measured in cell costimulated for 120 hours.

The results are presented in Table VI. The results indicate thatcostimulation with B7-2VIg leads to production of high levels of IL-2,IL-4, IFN-γ, and GM-CSF. Moreover, the amount of all 4 cytokinesproduced from T cells costimulated with B7-2VIg was higher than theamount of cytokines produced from T cells costimulated with B7-1Ig.Compared to B7-2Ig, B7-2VIg also costimulated the production of higheramounts of IL-2, IL-4 and GM-CSF and similar amounts of IFN-γ. Thus,B7-2VIg is a potent costimulator for production of cytokines by T cells.

Moreover, after 120 hours of culture, the T cells costimulated withB7-2VIg produced more than twice the amount of IL-4 produced by T cellscostimulated with B7-2Ig and approximately 8 fold the amount of IL-4produced by T cells costimulated with B7-1Ig. Thus, T cells costimulatedwith B7-2VIg fusion protein induces production of high levels of IL-4and this production is longlasting. Costimulation of T cells withB7-2VIg could thus drive T cells to a T helper 2 (Th2) state in longterm culture.

                  TABLE VI                                                        ______________________________________                                        Cytokine production by CD4+CD28+T cells costimulated with B7-2VIg,            B7-21g, B7-1Ig or anti-CD28 antibody                                                 IL-2    IL-2    IL-4  IL-4  IFN-g  GM-CSF                              costimulus                                                                           18 hr   120 hr  18 hr 120 hr                                                                              18 hr  18 hr                               ______________________________________                                        none    19      0       6.5  0     27.6    0                                  anti-CD28                                                                            2412    N.D.    83.2  0     28     25                                  B7-1Ig  734     0      10.4  5.7   27.8   10                                  B7-2Ig 1557    104     10.8  18.6  42.6   12                                  B7-2vIg                                                                              3073    262     29.8  40.8  37.5   30                                  ______________________________________                                    

M. B7-2Ig and B7-2VIg, but not B7-1Ig promote sustained T cell growth.

In this example, the capability of B7Ig fusion proteins to promotegrowth of T cells for extended periods was analysed.

CD28+ T lymphocytes were incubated in the presence of anti-CD3 plusanti-CD28, or B7-1Ig, B7-2Ig, or B7-2VIg immobilized on beads and thetotal cell numbers monitored over a period of 12 days. The results arepresented in FIG. 27. Cells stimulated with anti-CD3 alone fail toproliferate, and die. Cells stimulated with anti-CD3 plus B7-2Ig gothrough one cycle of replication and then apoptose. Cells stimulatedwith anti-CD3 plus anti-CD28 or B7-2Ig or B7-2vIg continue to replicate.Thus, B7-2Ig and B7-2VIg fusion proteins, but not B7-1Ig, are capable ofstimulating prolonged growth of CD28+ T cells.

EXAMPLE 8 Production and Characterization of Monoclonal Antibodies toHuman B7-2

A. Immunizations and Cell Fusions

Balb/c female mice (obtained from Taconic Labs, Germantown, N.Y.) wereimmunized intraperitoneally with 50 μg human B7.2-Ig emulsified incomplete Freund's adjuvant (Sigma Chemical Co., St. Louis, Mo.) or 10⁶CHO-human B7.2 cells per mouse. The mice were given two boosterimmunizations with 10-25 μg human B7.2-Ig emulsified in incompleteFreund's adjuvant (Sigma Chemical Co., St. Louis, Mo.) or CHO-human B7.2cells at fourteen day intervals following the initial immunization forthe next two months. The mice were bled by retro-orbital bleed and thesera assayed for the presence of antibodies reactive to the immunogen byELISA against human B7.2-Ig. ELISA against hCTLA4-Ig was also used tocontrol for Ig tail directed antibody responses. Mice showing a strongserological response were boosted intravenously via the tail vein with25 μg human hB7.2-Ig diluted in phosphate-buffered saline (PBS), pH 7.2(GIBCO, Grand Island, N.Y.). Three to four days following this boost,the spleens from these mice were fused 5:1 with SP 2/0 myeloma cells(American Type Culture Collection, Rockville, Md., No. CRL8006), whichare incapable of secreting both heavy and light immunoglobulin chains(Kearney et al. (1979) J. Immunol. 123:1548). Standard methods basedupon those developed by Kohler and Milstein (Nature (1975) 256:495) wereused.

B. Antibody Screening

After 10-21 days, supernatants from wells containing hybridoma coloniesfrom the fusion were screened for the presence of antibodies reactive tohuman B7.2 as follows: Each well of a 96 well flat bottomed plate(Costar Corp., Cat. #3590) was coated with 50 μl per well of a 1 μg/mlhuman B7.2-Ig solution or 5×10⁴ 3T3-hB7.2 cells on lysine coated platesin phosphate-buffered saline, pH 7.2, overnight at 4° C. The humanB7.2-Ig solution was aspirated off, or the cells were cross-linked tothe plates with glutaraldehyde, and the wells were washed three timeswith PBS, then blocked with 1% BSA solution (in PBS) (100 μl/well) forone hour at room temperature. Following this blocking incubation, thewells were washed three times with PBS and 50 μl of hybridomasupernatant was added per well and incubated for 1.5 hours at roomtemperature. Following this incubation, the wells were washed threetimes with PBS and then incubated for 1.5 hours at room temperature with50 μl per well of a 1:4000 dilution of horseradishperoxidase-conjugated, affinity purified, goat anti-mouse IgG or IgMheavy and light chain-specific antibodies (HRP; Zymed Laboratories, SanFrancisco, Calif.). The wells were then washed three times with PBS,followed by a 30 minute incubation in 50 μl per well of 1 mM2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in 0.1 MNa-Citrate, pH 4.2 to which a 1:1000 dilution of 30% hydrogen peroxidehad been added as a substrate for HRP to detect bound antibody. Theabsorbence was then determined at OD₄₁₀ on a spectrophotometricautoreader (Dynatech, Va.).

Three hybridomas, HA3.1F9, HA5.2B7 and HF2.3D1, were identified thatproduced antibodies to human B7.2-Ig. HA3.1F9 was determined to be ofthe IgGg 1 isotype, HA5.2B7 was determined to be of the IgG2b isotypeand HF2.3D1 as determined to be of the IgG2a isotype. Each of thesehybridomas were subcloned two additional times to insure that they weremonoclonal. Hybidoma cells were deposited with the American Type CultureCollection, which meets the requirements of the Budapest Treaty, on Jul.19, 1994 as ATCC Accession No. HB11688 (hybridoma HA3.1F9), ATCCAccession No. HB11687 (HA5.2B7) and ATCC Accession No. HB11686(HF2.3D1).

C. Competitive ELISA

Supernatants from the hybridomas HA3.1F9, HA5.2B7 and HF2.3D1 werefurther characterized by competitive ELISA, in which the ability of themonoclonal antibodies to inhibit the binding of biotinylated hCTLA4Ig toimmobilized hB7-2 immunoglobulin fusion proteins was examined.Biotinylation of hCTLA4Ig was performed using Pierce Immunopure NHS-LCBiotin (Cat. No. 21335). B7-2 immunoglobulin fusion proteins used were:hB7.2-Ig (full-length hB7-2), hB7.2-VIg (hB7-2 variable domain only) andhB7.2-CIg (B7-2 constant domain only). A hB7.1 -Ig fusion protein wasused as a control. For the ELISA, 96 well plates were coated with the Igfusion protein (50 μl/well of a 20 μg/ml solution) overnight at roomtemperature. The wells were washed three times with PBS, blocked with10% fetal bovine serum (FBS), 0.1% bovine serum albumin (BSA) in PBS for1 hour at room temperature, and washed again three times with PBS. Toeach well was added 50 μI of Bio-hCTLA4-Ig (70 ng/ml) and 50 μl ofcompetitor monoclonal antibody supernatant. Control antibodies were ananti-B7.1 mAb (EW3.5D12) and the anti-hB7-2 mAb B70 (IgG2biκ, obtainedfrom Pharmingen). The wells were washed again andstreptavidin-conjugated horse radish peroxidase (from Pierce, Cat. No.21126; 1:2000 dilution, 50 μl/well) was added and incubated for 30minutes at room temperature. The wells were washed again, followed by a30 minute incubation in 50 μl per well of ABTS in 0.1 M Na-Citrate, pH4.2 to which a 1:1000 dilution of 30% hydrogen peroxide had been addedas a substrate for HRP to detect bound antibody. The absorbence was thendetermined at OD₄₁₀ on a spectrophotometric autoreader (Dynatech, Va.).The results, shown in Table IV below, demonstrate that each of the mAbsproduced by the hybridomas HA3. IF9, HA5.2B7 and HF2.3D1 are able tocompetitively inhibit the binding of hCLTA4Ig to full-length hB7.2-Ig orhB7.2-VIg (hCTLA4Ig does not bind to hB7.2CIg).

                  TABLE IV                                                        ______________________________________                                                   Blocking of Binding                                                           hB7.1-Ig                                                                             hB7.2-Ig hB7.2-VIg                                                                              hB7.2-CIg                                 ______________________________________                                        EW3.5D12 (anti-hB7.1                                                                       Yes      No       No     No                                      mAb)                                                                          B70 (anti-hB7-2)                                                                           No       Yes      Yes    No                                      HA3.1F9 (anti-hB7-2)                                                                       No       Yes      Yes    No                                      HA5.2B7 (anti-hB7-2)                                                                       No       Yes      Yes    No                                      HF2.3D1 (anti-hB7-2)                                                                       No       Yes      Yes    No                                      ______________________________________                                    

D. Flow Cytometry

Supernatants from the hybridomas HA3.1F9, HA5.2B7 and HF2.3D1 were alsocharacterized by flow cytometry. Supernatants collected from the cloneswere screened by flow cytometry on CHO and 3T3 cells transfected toexpress hB7.2 (CHO-hB7.2 and 3T3-hB7.2, respectively) or controltransfected 3T3 cells (3T3-Neo). Flow cytometry was performed asfollows: 1×10⁶ cells were washed three times in 1% BSA in PBS, then thecells were incubated in 50 μl hybridoma supernatant or culture media per1¹⁰ ⁶ cells for 30 minutes at 4° C. Following the incubation, the cellswere washed three times with 1% BSA in PBS, then incubated in 50 μlfluorescein-conjugated goat anti-mouse IgG or IgM antibodies (ZymedLaboratories, San Francisco, Calif.) at 1:50 dilution per 1×10⁶ cellsfor 30 minutes at 4° C. The cells were then washed three times in 1% BSAin PBS and fixed with 1% paraformaldehyde solution. The cell sampleswere then analyzed on a FACScan flow cytometer (Becton Dickinson, SanJose Calif.). The results, shown in FIGS. 17, 18 and 19, demonstrate themonoclonal antibodies produced by the hybridomas HA3.1F9, HA5.2B7 andHF2.3D1 each bind to hB7-2 on the surface of cells.

E. Inhibition of Proliferation of Human T Cells by Anti-hB7-2 mAbs

Hybridoma supernatants containing anti-human B7-2 mAbs were tested fortheir ability to inhibit hB7-2 costimulation of human T cells. In thisassay, purified CD28⁺ human T cells were treated with submitogenicamounts of PMA (1 ng/ml) to deliver the primary signal and with CHOcells expressing hB7-2 on their surface to deliver the costimulatorysignal. Proliferation of the T cells was measured after three days inculture by the addition of ³ H-thymidine for the remaining 18 hours. Asshown in Table V, resting T cells show little proliferation as measuredby ³ H-thymidine incorporation (510 pm). Delivery of signal 1 by PMAresults in some proliferation (3800 pm) and T cells receiving both theprimary (PMA) and costimulatory (CHO/hB7-2) signals proliferatemaximally (9020 cpm). All three anti-hB7-2 mAbs tested reduce thecostimulatory signal induced proliferation to that found for PMA treatedcells alone showing that these mAbs can inhibit T cell proliferation byblocking the B7/CD28 costimulatory pathway.

                  TABLE V                                                         ______________________________________                                        Addition to CD28.sup.+  T Cells                                                                  hB7-2 mAb CPM                                              ______________________________________                                        --                 --        510                                              +PMA               --        3800                                             +PMA + CHO/hB7-2   --        9020                                             +PMA + CHO/hB7-2   HF2.301   3030                                             --                 HA5.2B7   1460                                             --                 HA3.1F9   2980                                             ______________________________________                                    

F. Antibodies to the B7-2 Variable domain block B7-2 function

In this example, the ability of a series of monoclonal antibodies toB7-2 to bind to the Ig-variable or Ig-constant domains of B7-2, an toinhibit T cell proliferation was analyzed.

Monoclonal antibodies to human B7-1 and B7-2 were prepared from Balb/cmice using SP2/0 cells and standard protocols. Briefly, Balb/c femalemice (Taconic Labs, Germantown, N.Y.) were immunized intraperitoneallywith either 50 μgs B7-2Ig emulsified in CFA (Sigma, St. Louis, Mo.) or10⁶ CHO/B7-2 cells. The mice were boosted twice at 14 day intervalsfollowing the initial immunization and once with B7-2Ig protein in PBS.Hybridoma colonies were established in 96 well tissue culture plates andthe culture supernatants were assayed for direct binding to B7-2Ig. AllmAbs were purified from ascites fluid on Protein-A sepharose asdescribed above. MAb B70 was purchased from PharMingen (San Diego,Calif.).

Purified mAbs were tested for their ability to bind to the various B7-Igforms as follows. Maxisorp plates (Nunc) were coated overnight at roomtemperature with 20 μg/ml of purified B7-2Ig protein in PBS. The plateswere then blocked with PBS/0.1% BSA for 1 h. Purified antibody (5 μgs/mlin PBS) was added to the test wells, the plates incubated for 1 hour,and then washed 5 times with PBS/0.05% Tween20. Goat anti-mouse IgG-HRP(Zymed) was added and allowed to react for 1 hour, followed by 5 washes.The plates were developed as described above.

The binding characteristics of the antibodies is indicated in Table VIIunder the heading "Recognition". As indicated in Table VII, allantibodies recognized B7-2Ig. Binding of the antibodies to B7-2VIg andB7-2CIg fusion proteins indicated that the antibodies recognize eitherthe variable region construct or the constant region construct, but notboth constructs.

The antibodies were further analyzed for their ability to inhibitbinding of B7-2Ig to CTLA4 and to CD28 and to inhibit T cellproliferation. Using a competition ELISA format, varying amounts of mAbswere added to wells coated B7-2Ig and containing 35 ngs/ml biotinylatedCTLA4Ig or CD28Ig. The ability of mAbs to disrupt the binding wasmeasured as a decrease in the specific signal of captured biotinylatedCTLA4Ig or CD28Ig. The capability of the antibodies to inhibitproliferation of T cells was determined by performing proliferationassays, as described above in which one of the antibodies was added.

The results are presented in Table VII under the heading "Inhibition".In general, antibodies to the V-domain of B7-2 inhibit binding of theB7-2Ig to CD28 and CTLA4 and also inhibit CHO/B7-2 driven T cellproliferation. Antibodies to the C-domain are not inhibitory suggestingthat the functionality of B7-2 resides in the V-domain.

                  TABLE VII                                                       ______________________________________                                        Characterization of anti-B7-2 antibodies                                               Recognition Inhibition                                                                B7v     B7c   CTLA4 CD28  T cell                             Anti-B7-2 mAbs                                                                           B7    domain  domain                                                                              binding                                                                             binding                                                                             prolif.                            ______________________________________                                        HA5.1F9                                                                              IgG1    +     -     -     +     +     +                                HA5.2B7                                                                              IgG2b   +     +     -     +     +     +                                HF2.3D11                                                                             IgG2a   +     +     -     +     +     +                                HF4.3C11                                                                             IgG1    +     +     -     +     +     +                                HF4.3E8                                                                              IgG1    +     +     -     +     +/-   +/-                              HF4.5B4                                                                              IgG1    +     -     +     nd    -     -                                HF4.5H12                                                                             IgM     +     -     +     -     -     -                                HF4.6B1                                                                              IgG2a   +     -     +     -     -     -                                HF4.6H4                                                                              IgG1    +     +     -     +     +     +                                B70    IgG2b   +     +     -     +     +     +                                ______________________________________                                    

Moreover, all of the anti-B7-1 and anti-B7-2 mAbs tested also recognizedtheir respective ligand when expressed on the surface of CHO cells andon the surface of activated human B cells where tested. None of theanti-B7-1 or anti-B7-2 mAbs showed any crossreactivity with the other B7protein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 55                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1120 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 107..1093                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - CACAGGGTGA AAGCTTTGCT TCTCTGCTGC TGTAACAGGG ACTAGCACAG AC - #ACACGGAT         60                                                                          #GAT CCC       115CAGAT ATTAGGTCAC AGCAGAAGCA GCCAAA ATG                      #Pro            Met Asp                                                       #                 1                                                           - CAG TGC ACT ATG GGA CTG AGT AAC ATT CTC TT - #T GTG ATG GCC TTC CTG          163                                                                          Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Ph - #e Val Met Ala Phe Leu           #      15                                                                     - CTC TCT GGT GCT GCT CCT CTG AAG ATT CAA GC - #T TAT TTC AAT GAG ACT          211                                                                          Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Al - #a Tyr Phe Asn Glu Thr           # 35                                                                          - GCA GAC CTG CCA TGC CAA TTT GCA AAC TCT CA - #A AAC CAA AGC CTG AGT          259                                                                          Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gl - #n Asn Gln Ser Leu Ser           #                 50                                                          - GAG CTA GTA GTA TTT TGG CAG GAC CAG GAA AA - #C TTG GTT CTG AAT GAG          307                                                                          Glu Leu Val Val Phe Trp Gln Asp Gln Glu As - #n Leu Val Leu Asn Glu           #             65                                                              - GTA TAC TTA GGC AAA GAG AAA TTT GAC AGT GT - #T CAT TCC AAG TAT ATG          355                                                                          Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Va - #l His Ser Lys Tyr Met           #         80                                                                  - GGC CGC ACA AGT TTT GAT TCG GAC AGT TGG AC - #C CTG AGA CTT CAC AAT          403                                                                          Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Th - #r Leu Arg Leu His Asn           #     95                                                                      - CTT CAG ATC AAG GAC AAG GGC TTG TAT CAA TG - #T ATC ATC CAT CAC AAA          451                                                                          Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cy - #s Ile Ile His His Lys           100                 1 - #05                 1 - #10                 1 -       #15                                                                           - AAG CCC ACA GGA ATG ATT CGC ATC CAC CAG AT - #G AAT TCT GAA CTG TCA          499                                                                          Lys Pro Thr Gly Met Ile Arg Ile His Gln Me - #t Asn Ser Glu Leu Ser           #               130                                                           - GTG CTT GCT AAC TTC AGT CAA CCT GAA ATA GT - #A CCA ATT TCT AAT ATA          547                                                                          Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Va - #l Pro Ile Ser Asn Ile           #           145                                                               - ACA GAA AAT GTG TAC ATA AAT TTG ACC TGC TC - #A TCT ATA CAC GGT TAC          595                                                                          Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Se - #r Ser Ile His Gly Tyr           #       160                                                                   - CCA GAA CCT AAG AAG ATG AGT GTT TTG CTA AG - #A ACC AAG AAT TCA ACT          643                                                                          Pro Glu Pro Lys Lys Met Ser Val Leu Leu Ar - #g Thr Lys Asn Ser Thr           #   175                                                                       - ATC GAG TAT GAT GGT ATT ATG CAG AAA TCT CA - #A GAT AAT GTC ACA GAA          691                                                                          Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gl - #n Asp Asn Val Thr Glu           180                 1 - #85                 1 - #90                 1 -       #95                                                                           - CTG TAC GAC GTT TCC ATC AGC TTG TCT GTT TC - #A TTC CCT GAT GTT ACG          739                                                                          Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Se - #r Phe Pro Asp Val Thr           #               210                                                           - AGC AAT ATG ACC ATC TTC TGT ATT CTG GAA AC - #T GAC AAG ACG CGG CTT          787                                                                          Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Th - #r Asp Lys Thr Arg Leu           #           225                                                               - TTA TCT TCA CCT TTC TCT ATA GAG CTT GAG GA - #C CCT CAG CCT CCC CCA          835                                                                          Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu As - #p Pro Gln Pro Pro Pro           #       240                                                                   - GAC CAC ATT CCT TGG ATT ACA GCT GTA CTT CC - #A ACA GTT ATT ATA TGT          883                                                                          Asp His Ile Pro Trp Ile Thr Ala Val Leu Pr - #o Thr Val Ile Ile Cys           #   255                                                                       - GTG ATG GTT TTC TGT CTA ATT CTA TGG AAA TG - #G AAG AAG AAG AAG CGG          931                                                                          Val Met Val Phe Cys Leu Ile Leu Trp Lys Tr - #p Lys Lys Lys Lys Arg           260                 2 - #65                 2 - #70                 2 -       #75                                                                           - CCT CGC AAC TCT TAT AAA TGT GGA ACC AAC AC - #A ATG GAG AGG GAA GAG          979                                                                          Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Th - #r Met Glu Arg Glu Glu           #               290                                                           - AGT GAA CAG ACC AAG AAA AGA GAA AAA ATC CA - #T ATA CCT GAA AGA TCT         1027                                                                          Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile Hi - #s Ile Pro Glu Arg Ser           #           305                                                               - GAT GAA GCC CAG CGT GTT TTT AAA AGT TCG AA - #G ACA TCT TCA TGC GAC         1075                                                                          Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Ly - #s Thr Ser Ser Cys Asp           #       320                                                                   - AAA AGT GAT ACA TGT TTT TAATTAAAGA GTAAAGCCCA AA - #AAAAA                   1120                                                                          Lys Ser Asp Thr Cys Phe                                                           325                                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 329 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Asp Pro Gln Cys Thr Met Gly Leu Ser As - #n Ile Leu Phe Val Met         #                 15                                                          - Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Ly - #s Ile Gln Ala Tyr Phe         #             30                                                              - Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Al - #a Asn Ser Gln Asn Gln         #         45                                                                  - Ser Leu Ser Glu Leu Val Val Phe Trp Gln As - #p Gln Glu Asn Leu Val         #     60                                                                      - Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Ph - #e Asp Ser Val His Ser         # 80                                                                          - Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser As - #p Ser Trp Thr Leu Arg         #                 95                                                          - Leu His Asn Leu Gln Ile Lys Asp Lys Gly Le - #u Tyr Gln Cys Ile Ile         #           110                                                               - His His Lys Lys Pro Thr Gly Met Ile Arg Il - #e His Gln Met Asn Ser         #       125                                                                   - Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pr - #o Glu Ile Val Pro Ile         #   140                                                                       - Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Le - #u Thr Cys Ser Ser Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - His Gly Tyr Pro Glu Pro Lys Lys Met Ser Va - #l Leu Leu Arg Thr Lys         #               175                                                           - Asn Ser Thr Ile Glu Tyr Asp Gly Ile Met Gl - #n Lys Ser Gln Asp Asn         #           190                                                               - Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Le - #u Ser Val Ser Phe Pro         #       205                                                                   - Asp Val Thr Ser Asn Met Thr Ile Phe Cys Il - #e Leu Glu Thr Asp Lys         #   220                                                                       - Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Gl - #u Leu Glu Asp Pro Gln         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Pro Pro Asp His Ile Pro Trp Ile Thr Al - #a Val Leu Pro Thr Val         #               255                                                           - Ile Ile Cys Val Met Val Phe Cys Leu Ile Le - #u Trp Lys Trp Lys Lys         #           270                                                               - Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gl - #y Thr Asn Thr Met Glu         #       285                                                                   - Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Gl - #u Lys Ile His Ile Pro         #   300                                                                       - Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Ly - #s Ser Ser Lys Thr Ser         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Cys Asp Lys Ser Asp Thr Cys Phe                                                         325                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 # 20               AGGG                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #  18              AG                                                         - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #21                GATC C                                                     - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #21                TGGG C                                                     - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #21                ATTC G                                                     - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #21                CTGA G                                                     - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #21                GCAG C                                                     - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #21                ACGC G                                                     - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #21                GAGA G                                                     - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #21                CATT G                                                     - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #21                CGTC G                                                     - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #21                GAGT G                                                     - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 8 base p - #airs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #           8                                                                 - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Lys Tyr Met Gly Arg Thr Ser Phe Asp                                         #5                                                                            - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Lys Ser Gln Asp Asn Val Thr Glu Lys Tyr As - #p Val Ser                     #10                                                                           - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Trp Lys Trp Lys Lys Lys Lys Arg Pro Arg As - #n Ser Tyr Lys Cys             #15                                                                           - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1163 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 111..1040                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - CCCACGCGTC CGGGAGCAAG CAGACGCGTA AGAGTGGCTC CTGTAGGCAG CA - #CGGACTTG         60                                                                          - AACAACCAGA CTCCTGTAGA CGTGTTCCAG AACTTACGGA AGCACCCACG AT - #G GAC           116                                                                          # Met Asp                                                                     #   1                                                                         - CCC AGA TGC ACC ATG GGC TTG GCA ATC CTT AT - #C TTT GTG ACA GTC TTG          164                                                                          Pro Arg Cys Thr Met Gly Leu Ala Ile Leu Il - #e Phe Val Thr Val Leu           #          15                                                                 - CTG ATC TCA GAT GCT GTT TCC GTG GAG ACG CA - #A GCT TAT TTC AAT GGG          212                                                                          Leu Ile Ser Asp Ala Val Ser Val Glu Thr Gl - #n Ala Tyr Phe Asn Gly           #     30                                                                      - ACT GCA TAT CTG CCG TGC CCA TTT ACA AAG GC - #T CAA AAC ATA AGC CTG          260                                                                          Thr Ala Tyr Leu Pro Cys Pro Phe Thr Lys Al - #a Gln Asn Ile Ser Leu           # 50                                                                          - AGT GAG CTG GTA GTA TTT TGG CAG GAC CAG CA - #A AAG TTG GTT CTG TAC          308                                                                          Ser Glu Leu Val Val Phe Trp Gln Asp Gln Gl - #n Lys Leu Val Leu Tyr           #                 65                                                          - GAG CAC TAT TTG GGC ACA GAG AAA CTT GAT AG - #T GTG AAT GCC AAG TAC          356                                                                          Glu His Tyr Leu Gly Thr Glu Lys Leu Asp Se - #r Val Asn Ala Lys Tyr           #             80                                                              - CTG GGC CGC ACG AGC TTT GAC AGG AAC AAC TG - #G ACT CTA CGA CTT CAC          404                                                                          Leu Gly Arg Thr Ser Phe Asp Arg Asn Asn Tr - #p Thr Leu Arg Leu His           #         95                                                                  - AAT GTT CAG ATC AAG GAC ATG GGC TCG TAT GA - #T TGT TTT ATA CAA AAA          452                                                                          Asn Val Gln Ile Lys Asp Met Gly Ser Tyr As - #p Cys Phe Ile Gln Lys           #   110                                                                       - AAG CCA CCC ACA GGA TCA ATT ATC CTC CAA CA - #G ACA TTA ACA GAA CTG          500                                                                          Lys Pro Pro Thr Gly Ser Ile Ile Leu Gln Gl - #n Thr Leu Thr Glu Leu           115                 1 - #20                 1 - #25                 1 -       #30                                                                           - TCA GTG ATC GCC AAC TTC AGT GAA CCT GAA AT - #A AAA CTG GCT CAG AAT          548                                                                          Ser Val Ile Ala Asn Phe Ser Glu Pro Glu Il - #e Lys Leu Ala Gln Asn           #               145                                                           - GTA ACA GGA AAT TCT GGC ATA AAT TTG ACC TG - #C ACG TCT AAG CAA GGT          596                                                                          Val Thr Gly Asn Ser Gly Ile Asn Leu Thr Cy - #s Thr Ser Lys Gln Gly           #           160                                                               - CAC CCG AAA CCT AAG AAG ATG TAT TTT CTG AT - #A ACT AAT TCA ACT AAT          644                                                                          His Pro Lys Pro Lys Lys Met Tyr Phe Leu Il - #e Thr Asn Ser Thr Asn           #       175                                                                   - GAG TAT GGT GAT AAC ATG CAG ATA TCA CAA GA - #T AAT GTC ACA GAA CTG          692                                                                          Glu Tyr Gly Asp Asn Met Gln Ile Ser Gln As - #p Asn Val Thr Glu Leu           #   190                                                                       - TTC AGT ATC TCC AAC AGC CTC TCT CTT TCA TT - #C CCG GAT GGT GTG TGG          740                                                                          Phe Ser Ile Ser Asn Ser Leu Ser Leu Ser Ph - #e Pro Asp Gly Val Trp           195                 2 - #00                 2 - #05                 2 -       #10                                                                           - CAT ATG ACC GTT GTG TGT GTT CTG GAA ACG GA - #G TCA ATG AAG ATT TCC          788                                                                          His Met Thr Val Val Cys Val Leu Glu Thr Gl - #u Ser Met Lys Ile Ser           #               225                                                           - TCC AAA CCT CTC AAT TTC ACT CAA GAG TTT CC - #A TCT CCT CAA ACG TAT          836                                                                          Ser Lys Pro Leu Asn Phe Thr Gln Glu Phe Pr - #o Ser Pro Gln Thr Tyr           #           240                                                               - TGG AAG GAG ATT ACA GCT TCA GTT ACT GTG GC - #C CTC CTC CTT GTG ATG          884                                                                          Trp Lys Glu Ile Thr Ala Ser Val Thr Val Al - #a Leu Leu Leu Val Met           #       255                                                                   - CTG CTC ATC ATT GTA TGT CAC AAG AAG CCG AA - #T CAG CCT AGC AGG CCC          932                                                                          Leu Leu Ile Ile Val Cys His Lys Lys Pro As - #n Gln Pro Ser Arg Pro           #   270                                                                       - AGC AAC ACA GCC TCT AAG TTA GAG CGG GAT AG - #T AAC GCT GAC AGA GAG          980                                                                          Ser Asn Thr Ala Ser Lys Leu Glu Arg Asp Se - #r Asn Ala Asp Arg Glu           275                 2 - #80                 2 - #85                 2 -       #90                                                                           - ACT ATC AAC CTG AAG GAA CTT GAA CCC CAA AT - #T GCT TCA GCA AAA CCA         1028                                                                          Thr Ile Asn Leu Lys Glu Leu Glu Pro Gln Il - #e Ala Ser Ala Lys Pro           #               305                                                           - AAT GCA GAG TGAAGGCAGT GAGAGCCTGA GGAAAGAGTT AAAAATTGC - #T                 1077                                                                          Asn Ala Glu                                                                   TTGCCTGAAA TAAGAAGTGC AGAGTTTCTC AGAATTCAAA AATGTTCTCA GC - #TGATTGGA         1137                                                                          #            1163  ATTA AAGAAC                                                - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 309 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Met Asp Pro Arg Cys Thr Met Gly Leu Ala Il - #e Leu Ile Phe Val Thr         #                 15                                                          - Val Leu Leu Ile Ser Asp Ala Val Ser Val Gl - #u Thr Gln Ala Tyr Phe         #             30                                                              - Asn Gly Thr Ala Tyr Leu Pro Cys Pro Phe Th - #r Lys Ala Gln Asn Ile         #         45                                                                  - Ser Leu Ser Glu Leu Val Val Phe Trp Gln As - #p Gln Gln Lys Leu Val         #     60                                                                      - Leu Tyr Glu His Tyr Leu Gly Thr Glu Lys Le - #u Asp Ser Val Asn Ala         # 80                                                                          - Lys Tyr Leu Gly Arg Thr Ser Phe Asp Arg As - #n Asn Trp Thr Leu Arg         #                 95                                                          - Leu His Asn Val Gln Ile Lys Asp Met Gly Se - #r Tyr Asp Cys Phe Ile         #           110                                                               - Gln Lys Lys Pro Pro Thr Gly Ser Ile Ile Le - #u Gln Gln Thr Leu Thr         #       125                                                                   - Glu Leu Ser Val Ile Ala Asn Phe Ser Glu Pr - #o Glu Ile Lys Leu Ala         #   140                                                                       - Gln Asn Val Thr Gly Asn Ser Gly Ile Asn Le - #u Thr Cys Thr Ser Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Gly His Pro Lys Pro Lys Lys Met Tyr Ph - #e Leu Ile Thr Asn Ser         #               175                                                           - Thr Asn Glu Tyr Gly Asp Asn Met Gln Ile Se - #r Gln Asp Asn Val Thr         #           190                                                               - Glu Leu Phe Ser Ile Ser Asn Ser Leu Ser Le - #u Ser Phe Pro Asp Gly         #       205                                                                   - Val Trp His Met Thr Val Val Cys Val Leu Gl - #u Thr Glu Ser Met Lys         #   220                                                                       - Ile Ser Ser Lys Pro Leu Asn Phe Thr Gln Gl - #u Phe Pro Ser Pro Gln         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Tyr Trp Lys Glu Ile Thr Ala Ser Val Th - #r Val Ala Leu Leu Leu         #               255                                                           - Val Met Leu Leu Ile Ile Val Cys His Lys Ly - #s Pro Asn Gln Pro Ser         #           270                                                               - Arg Pro Ser Asn Thr Ala Ser Lys Leu Glu Ar - #g Asp Ser Asn Ala Asp         #       285                                                                   - Arg Glu Thr Ile Asn Leu Lys Glu Leu Glu Pr - #o Gln Ile Ala Ser Ala         #   300                                                                       - Lys Pro Asn Ala Glu                                                         305                                                                           - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #21                GCTG G                                                     - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #21                CAAG G                                                     - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #21                AATA C                                                     - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #21                CAGT G                                                     - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1491 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #en                                                 (F) TISSUE TYPE: lympho - #id                                                 (G) CELL TYPE: B cel - #l                                                     (H) CELL LINE: Raji                                                 -    (vii) IMMEDIATE SOURCE:                                                  #pCDM8 vector LIBRARY: cDNA in                                                #clone #13(B) CLONE: B7, Raji                                                 -   (viii) POSITION IN GENOME:                                                          (A) CHROMOSOME/SEGMENT: 3                                           -     (ix) FEATURE:                                                           #reading frame (translated region)                                                      (B) LOCATION:  318 t - #o 1181 bp                                             (C) IDENTIFICATION METHOD: - # similarity to other pattern          -     (ix) FEATURE:                                                                     (A) NAME/KEY:  Alternat - #e polyadenylation signal                 #to 1479 bpB) LOCATION:  1474                                                           (C) IDENTIFICATION METHOD: - # similarity to other pattern          -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: FREEMAN, G - #ORDON J.                                 #ARNOLD S.     FREEDMAN,                                                                     SEGIL, JE - #FFREY M.                                                         LEE, GRAC - #E                                                 #JAMES F.      WHITMAN,                                                                      NADLER, L - #EE M.                                             #New Member Of The Ig Superfamily With                                                       Unique Ex - #pression On Activated And Neoplastic B Cells                (C) JOURNAL: The Journa - #l of Immunology                                    (D) VOLUME: 143                                                               (E) ISSUE: 8                                                                  (F) PAGES: 2714-2722                                                          (G) DATE: 15-OCT-1989                                                         (H) RELEVANT RESIDUES I - #N SEQ ID NO:28: FROM 1 TO 1491           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - CCAAAGAAAA AGTGATTTGT CATTGCTTTA TAGACTGTAA GAAGAGAACA TC - #TCAGAAGT         60                                                                          - GGAGTCTTAC CCTGAAATCA AAGGATTTAA AGAAAAAGTG GAATTTTTCT TC - #AGCAAGCT        120                                                                          - GTGAAACTAA ATCCACAACC TTTGGAGACC CAGGAACACC CTCCAATCTC TG - #TGTGTTTT        180                                                                          - GTAAACATCA CTGGAGGGTC TTCTACGTGA GCAATTGGAT TGTCATCAGC CC - #TGCCTGTT        240                                                                          - TTGCACCTGG GAAGTGCCCT GGTCTTACTT GGGTCCAAAT TGTTGGCTTT CA - #CTTTTGAC        300                                                                          #GGA ACA TCA CCA TCC   353GC CAC ACA CGG AGG CAG                              #Pro Sery His Thr Arg Arg Gln Gly Thr Ser                                     25 - #                                                                        - AAG TGT CCA TAC CTG AAT TTC TTT CAG CTC TT - #G GTG CTG GCT GGT CTT          401                                                                          Lys Cys Pro Tyr Leu Asn Phe Phe Gln Leu Le - #u Val Leu Ala Gly Leu           10                                                                            - TCT CAC TTC TGT TCA GGT GTT ATC CAC GTG AC - #C AAG GAA GTG AAA GAA          449                                                                          Ser His Phe Cys Ser Gly Val Ile His Val Th - #r Lys Glu Val Lys Glu           #   10                                                                        - GTG GCA ACG CTG TCC TGT GGT CAC AAT GTT TC - #T GTT GAA GAG CTG GCA          497                                                                          Val Ala Thr Leu Ser Cys Gly His Asn Val Se - #r Val Glu Glu Leu Ala           #                 25                                                          - CAA ACT CGC ATC TAC TGG CAA AAG GAG AAG AA - #A ATG GTG CTG ACT ATG          545                                                                          Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Ly - #s Met Val Leu Thr Met           #             40                                                              - ATG TCT GGG GAC ATG AAT ATA TGG CCC GAG TA - #C AAG AAC CGG ACC ATC          593                                                                          Met Ser Gly Asp Met Asn Ile Trp Pro Glu Ty - #r Lys Asn Arg Thr Ile           #         55                                                                  - TTT GAT ATC ACT AAT AAC CTC TCC ATT GTG AT - #C CTG GCT CTG CGC CCA          641                                                                          Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Il - #e Leu Ala Leu Arg Pro           #     70                                                                      - TCT GAC GAG GGC ACA TAC GAG TGT GTT GTT CT - #G AAG TAT GAA AAA GAC          689                                                                          Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Le - #u Lys Tyr Glu Lys Asp           # 90                                                                          - GCT TTC AAG CGG GAA CAC CTG GCT GAA GTG AC - #G TTA TCA GTC AAA GCT          737                                                                          Ala Phe Lys Arg Glu His Leu Ala Glu Val Th - #r Leu Ser Val Lys Ala           #                105                                                          - GAC TTC CCT ACA CCT AGT ATA TCT GAC TTT GA - #A ATT CCA ACT TCT AAT          785                                                                          Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Gl - #u Ile Pro Thr Ser Asn           #           120                                                               - ATT AGA AGG ATA ATT TGC TCA ACC TCT GGA GG - #T TTT CCA GAG CCT CAC          833                                                                          Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gl - #y Phe Pro Glu Pro His           #       135                                                                   - CTC TCC TGG TTG GAA AAT GGA GAA GAA TTA AA - #T GCC ATC AAC ACA ACA          881                                                                          Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu As - #n Ala Ile Asn Thr Thr           #   150                                                                       - GTT TCC CAA GAT CCT GAA ACT GAG CTC TAT GC - #T GTT AGC AGC AAA CTG          929                                                                          Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Al - #a Val Ser Ser Lys Leu           155                 1 - #60                 1 - #65                 1 -       #70                                                                           - GAT TTC AAT ATG ACA ACC AAC CAC AGC TTC AT - #G TGT CTC ATC AAG TAT          977                                                                          Asp Phe Asn Met Thr Thr Asn His Ser Phe Me - #t Cys Leu Ile Lys Tyr           #               185                                                           - GGA CAT TTA AGA GTG AAT CAG ACC TTC AAC TG - #G AAT ACA ACC AAG CAA         1025                                                                          Gly His Leu Arg Val Asn Gln Thr Phe Asn Tr - #p Asn Thr Thr Lys Gln           #           200                                                               - GAG CAT TTT CCT GAT AAC CTG CTC CCA TCC TG - #G GCC ATT ACC TTA ATC         1073                                                                          Glu His Phe Pro Asp Asn Leu Leu Pro Ser Tr - #p Ala Ile Thr Leu Ile           #       215                                                                   - TCA GTA AAT GGA ATT TTT GTG ATA TGC TGC CT - #G ACC TAC TGC TTT GCC         1121                                                                          Ser Val Asn Gly Ile Phe Val Ile Cys Cys Le - #u Thr Tyr Cys Phe Ala           #   230                                                                       - CCA AGA TGC AGA GAG AGA AGG AGG AAT GAG AG - #A TTG AGA AGG GAA AGT         1169                                                                          Pro Arg Cys Arg Glu Arg Arg Arg Asn Glu Ar - #g Leu Arg Arg Glu Ser           235                 2 - #40                 2 - #45                 2 -       #50                                                                           #AAGATCTGAA        1221AGTGTC CGCAGAAGCA AGGGGCTGAA                           Val Arg Pro Val                                                               - GGTAGCCTCC GTCATCTCTT CTGGGATACA TGGATCGTGG GGATCATGAG GC - #ATTCTTCC       1281                                                                          - CTTAACAAAT TTAAGCTGTT TTACCCACTA CCTCACCTTC TTAAAAACCT CT - #TTCAGATT       1341                                                                          - AAGCTGAACA GTTACAAGAT GGCTGGCATC CCTCTCCTTT CTCCCCATAT GC - #AATTTGCT       1401                                                                          - TAATGTAACC TCTTCTTTTG CCATGTTTCC ATTCTGCCAT CTTGAATTGT CT - #TGTCAGCC       1461                                                                          #         1491     AACA CTAATTTGAG                                            - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 288 amino                                                         (B) TYPE: amino acid                                                          (C) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                                       (A) DESCRIPTION: B cell - # activation antigen; natural ligand      #T cell surface antigen; transmembrane protein                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: signal se - #quence                                   #-1to     (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # amino terminal sequencing of                        soluble p - #rotein                                            #hydrophobic) OTHER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: extracellula - #r domain                                        (B) LOCATION: 1 to 2 - #08                                                    (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: transmembran - #e domain                              #235      (B) LOCATION: 209 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: intracellula - #r domain                              #254      (B) LOCATION: 236 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #glycosylationNAME/KEY: N-linked                                              #21       (B) LOCATION: 19 to                                                           (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #glycosylationNAME/KEY: N-linked                                              #57       (B) LOCATION: 55 to                                                           (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #glycosylationNAME/KEY: N-linked                                              #66       (B) LOCATION: 64 to                                                           (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #glycosylationNAME/KEY: N-linked                                              #154      (B) LOCATION: 152 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #glycosylationNAME/KEY: N-linked                                              #175      (B) LOCATION: 173 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #glycosylationNAME/KEY: N-linked                                              #179      (B) LOCATION: 177 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #glycosylationNAME/KEY: N-linked                                              #194      (B) LOCATION: 192 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #glycosylationNAME/KEY: N-linked                                              #200      (B) LOCATION: 198 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #domain   (A) NAME/KEY: Ig V-set                                                        (B) LOCATION: 1 to 1 - #04                                                    (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #domain   (A) NAME/KEY: Ig C-set                                                        (B) LOCATION:  105 t - #o 202                                                 (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: FREEMAN, G - #ORDON J.                                 #ARNOLD S.     FREEDMAN,                                                                     SEGIL, JE - #FFREY M.                                                         LEE, GRAC - #E                                                 #JAMES F.      WHITMAN,                                                                      NADLER, L - #EE M.                                                       (B) TITLE: B7, A New - # Member Of The Ig Superfamily With                         Unique Ex - #pression On Activated And Neoplastic B Cells                (C) JOURNAL: The Journa - #l of Immunology                                    (D) VOLUME: 143                                                               (E) ISSUE: 8                                                                  (F) PAGES: 2714-2722                                                          (G) DATE:  15-OCT-1989                                              26 to 262 (H) RELEVANT RESIDUES I - #N SEQ ID NO:29: From                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Met Gly His Thr Arg Arg Gln Gly Thr Ser Pr - #o Ser Lys Cys Pro Tyr         20                                                                            - Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gl - #y Leu Ser His Phe Cys         - Ser Gly Val Ile His Val Thr Lys Glu Val Ly - #s Glu Val Ala Thr Leu         #     10                                                                      - Ser Cys Gly His Asn Val Ser Val Glu Glu Le - #u Ala Gln Thr Arg Ile         # 30                                                                          - Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Th - #r Met Met Ser Gly Asp         #                 45                                                          - Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Th - #r Ile Phe Asp Ile Thr         #             60                                                              - Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Ar - #g Pro Ser Asp Glu Gly         #         75                                                                  - Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Ly - #s Asp Ala Phe Lys Arg         #     90                                                                      - Glu His Leu Ala Glu Val Thr Leu Ser Val Ly - #s Ala Asp Phe Pro Thr         #110                                                                          - Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Se - #r Asn Ile Arg Arg Ile         #               125                                                           - Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pr - #o His Leu Ser Trp Leu         #           140                                                               - Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Th - #r Thr Val Ser Gln Asp         #       155                                                                   - Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Ly - #s Leu Asp Phe Asn Met         #   170                                                                       - Thr Thr Asn His Ser Phe Met Cys Leu Ile Ly - #s Tyr Gly His Leu Arg         175                 1 - #80                 1 - #85                 1 -       #90                                                                           - Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Ly - #s Gln Glu His Phe Pro         #               205                                                           - Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Le - #u Ile Ser Val Asn Gly         #           220                                                               - Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Ph - #e Ala Pro Arg Cys Arg         #       235                                                                   - Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Gl - #u Ser Val Arg Pro Val         #   250                                                                       - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1716 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -    (iii) HYPOTHETICAL: no                                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Mus muscu - #lus                                      #germ line(D) DEVELOPMENTAL STAGE:                                                      (F) TISSUE TYPE: lympho - #id                                                 (G) CELL TYPE: B lym - #phocyte                                               (H) CELL LINE: 70Z a - #nd A20                                      -    (vii) IMMEDIATE SOURCE:                                                  #pCDM8 vector LIBRARY: cDNA in                                                #s 1 and 29B) CLONE: B7 #'                                                    -     (ix) FEATURE:                                                           #region   (A) NAME/KEY: translated                                            #1166 bp  (B) LOCATION: 249 to                                                          (C) IDENTIFICATION METHOD: - # similarity to other pattern          -     (ix) FEATURE:                                                           #ATG initiation codons: Alternate                                             #227 and 270 to 272ION: 225 to                                                          (C) IDENTIFICATION METHOD: - # similarity to other pattern          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - GAGTTTTATA CCTCAATAGA CTCTTACTAG TTTCTCTTTT TCAGGTTGTG AA - #ACTCAACC         60                                                                          - TTCAAAGACA CTCTGTTCCA TTTCTGTGGA CTAATAGGAT CATCTTTAGC AT - #CTGCCGGG        120                                                                          - TGGATGCCAT CCAGGCTTCT TTTTCTACAT CTCTGTTTCT CGATTTTTGT GA - #GCCTAGGA        180                                                                          - GGTGCCTAAG CTCCATTGGC TCTAGATTCC TGGCTTTCCC CATCATGTTC TC - #CAAAGCAT        240                                                                          #GAT ACA CCA CTC CTC     290GT CAG TTG ATG CAG                                         Met Ala Cys Asn Cys G - #ln Leu Met Gln Asp Thr Pro Leu Leu          25                                                                            - AAG TTT CCA TGT CCA AGG CTC AAT CTT CTC TT - #T GTG CTG CTG ATT CGT          338                                                                          Lys Phe Pro Cys Pro Arg Leu Ile Leu Leu Ph - #e Val Leu Leu Ile Arg           10                                                                            - CTT TCA CAA GTG TCT TCA GAT GTT GAT GAA CA - #A CTG TCC AAG TCA GTG          386                                                                          Leu Ser Gln Val Ser Ser Asp Val Asp Glu Gl - #n Leu Ser Lys Ser Val           #      5                                                                      - AAA GAT AAG GTA TTG CTG CCT TGC CGT TAC AA - #C TCT CCT CAT GAA GAT          434                                                                          Lys Asp Lys Val Leu Leu Pro Cys Arg Tyr As - #n Ser Pro His Glu Asp           # 25                                                                          - GAG TCT GAA GAC CGA ATC TAC TGG CAA AAA CA - #T GAC AAA GTG GTG CTG          482                                                                          Glu Ser Glu Asp Arg Ile Tyr Trp Gln Lys Hi - #s Asp Lys Val Val Leu           #                 40                                                          - TCT GTC ATT GCT GGG AAA CTA AAA GTG TGG CC - #C GAG TAT AAG AAC CGG          530                                                                          Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pr - #o Glu Tyr Lys Asn Arg           #             55                                                              - ACT TTA TAT GAC AAC ACT ACC TAC TCT CTT AT - #C ATC CTG GGC CTG GTC          578                                                                          Thr Leu Tyr Asp Asn Thr Thr Tyr Ser Leu Il - #e Ile Leu Gly Leu Val           #         70                                                                  - CTT TCA GAC CGG GGC ACA TAC AGC TGT GTC GT - #T CAA AAG AAG GAA AGA          626                                                                          Leu Ser Asp Arg Gly Thr Tyr Ser Cys Val Va - #l Gln Lys Lys Glu Arg           #     85                                                                      - GGA ACG TAT GAA GTT AAA CAC TTG GCT TTA GT - #A AAG TTG TCC ATC AAA          674                                                                          Gly Thr Tyr Glu Val Lys His Leu Ala Leu Va - #l Lys Leu Ser Ile Lys           #105                                                                          - GCT GAC TTC TCT ACC CCC AAC ATA ACT GAG TC - #T GGA AAC CCA TCT GCA          722                                                                          Ala Asp Phe Ser Thr Pro Asn Ile Thr Glu Se - #r Gly Asn Pro Ser Ala           #               120                                                           - GAC ACT AAA AGG ATT ACC TGC TTT GCT TCC GG - #G GGT TTC CCA AAG CCT          770                                                                          Asp Thr Lys Arg Ile Thr Cys Phe Ala Ser Gl - #y Gly Phe Pro Lys Pro           #           135                                                               - CGC TTC TCT TGG TTG GAA AAT GGA AGA GAA TT - #A CCT GGC ATC AAT ACG          818                                                                          Arg Phe Ser Trp Leu Glu Asn Gly Arg Glu Le - #u Pro Gly Ile Asn Thr           #       150                                                                   - ACA ATT TCC CAG GAT CCT GAA TCT GAA TTG TA - #C ACC ATT AGT AGC CAA          866                                                                          Thr Ile Ser Gln Asp Pro Glu Ser Glu Leu Ty - #r Thr Ile Ser Ser Gln           #   165                                                                       - CTA GAT TTC AAT ACG ACT CGC AAC CAC ACC AT - #T AAG TGT CTC ATT AAA          914                                                                          Leu Asp Phe Asn Thr Thr Arg Asn His Thr Il - #e Lys Cys Leu Ile Lys           170                 1 - #75                 1 - #80                 1 -       #85                                                                           - TAT GGA GAT GCT CAC GTG TCA GAG GAC TTC AC - #C TGG GAA AAA CCC CCA          962                                                                          Tyr Gly Asp Ala His Val Ser Glu Asp Phe Th - #r Trp Glu Lys Pro Pro           #               200                                                           - GAA GAC CCT CCT GAT AGC AAG AAC ACA CTT GT - #G CTC TTT GGG GCA GGA         1010                                                                          Glu Asp Pro Pro Asp Ser Lys Asn Thr Leu Va - #l Leu Phe Gly Ala Gly           #           215                                                               - TTC GGC GCA GTA ATA ACA GTC GTC GTC ATC GT - #T GTC ATC ATC AAA TGC         1058                                                                          Phe Gly Ala Val Ile Thr Val Val Val Ile Va - #l Val Ile Ile Lys Cys           #       230                                                                   - TTC TGT AAG CAC AGA AGC TGT TTC AGA AGA AA - #T GAG GCA AGC AGA GAA         1106                                                                          Phe Cys Lys His Arg Ser Cys Phe Arg Arg As - #n Glu Ala Ser Arg Glu           #   245                                                                       - ACA AAC AAC AGC CTT ACC TTC GGG CCT GAA GA - #A GCA TTA GCT GAA CAG         1154                                                                          Thr Asn Asn Ser Leu Thr Phe Gly Pro Glu Gl - #u Ala Leu Ala Glu Gln           250                 2 - #55                 2 - #60                 2 -       #65                                                                           - ACC GTC TTC CTT TAGTTCTTCT CTGTCCATGT GGGATACATG GT - #ATTATGTG             1206                                                                          Thr Val Phe Leu                                                               - GCTCATGAGG TACAATCTTT CTTTCAGCAC CGTGCTAGCT GATCTTTCGG AC - #AACTTGAC       1266                                                                          - ACAAGATAGA GTTAACTGGG AAGAGAAAGC CTTGAATGAG GATTTCTTTC CA - #TCAGGAAG       1326                                                                          - CTACGGGCAA GTTTGCTGGG CCTTTGATTG CTTGATGACT GAAGTGGAAA GG - #CTGAGCCC       1386                                                                          - ACTGTGGGTG GTGCTAGCCC TGGGCAGGGG CAGGTGACCC TGGGTGGTAT AA - #GAAAAAGA       1446                                                                          - GCTGTCACTA AAAGGAGAGG TGCCTAGTCT TACTGCAACT TGATATGTCA TG - #TTTGGTTG       1506                                                                          - GTGTCTGTGG GAGGCCTGCC CTTTTCTGAA GAGAAGTGGT GGGAGAGTGG AT - #GGGGTGGG       1566                                                                          - GGCAGAGGAA AAGTGGGGGA GAGGGCCTGG GAGGAGAGGA GGGAGGGGGA CG - #GGGTGGGG       1626                                                                          - GTGGGGAAAA CTATGGTTGG GATGTAAAAA CGGATAATAA TATAAATATT AA - #ATAAAAAG       1686                                                                          #         1716     AAAA AAAAAAAAAA                                            - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 306 amino                                                         (B) TYPE: amino acid                                                          (C) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                                       (A) DESCRIPTION: B lymp - #hocyte activation antigen; Ig            #member; T cell costimulatory signal                                                         via activ - #ation of CD28 pathways, binds to CD28+            #transmembrane protein,                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: signal se - #quence                                   #-1to     (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       #hydrophobic) OTHER INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: extracellula - #r domain                                        (B) LOCATION: 1 to 2 - #10                                                    (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: transmembran - #e domain                              #235      (B) LOCATION: 211 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: intracellula - #r (cytoplasmic) domain                #269      (B) LOCATION: 236 to                                                          (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #domain   (A) NAME/KEY: Ig V-set                                                        (B) LOCATION: 1 to 1 - #05                                                    (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -     (ix) FEATURE:                                                           #domain   (A) NAME/KEY: Ig C-set                                                        (B) LOCATION:  106 t - #o 199                                                 (C) IDENTIFICATION METHOD: - # similarity with known                               sequence                                                       -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: FREEMAN, G - #ORDON J.                                                GRAY, GAR - #Y S.                                                             GIMMI, CL - #AUDE D.                                           #DAVID B.      LOMBARD,                                                                      ZHOU, LIA - #NG-JI                                                            WHITE, MI - #CHAEL                                             #JOYCE D.      FINGEROTH,                                                     #JOHN G.       GRIBBEN,                                                                      NADLER, L - #EE M.                                                       (B) TITLE: Structure, E - #xpression, and T Cell Costimulatory      #Of The Murine Homologue Of The Human B                                       #Activation Antigen B7yte                                                               (C) JOURNAL: Journal of - # Experimental Medicine                             (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE: IN PRESS                                                  37 to 269 (H) RELEVANT RESIDUES I - #N SEQ ID NO:31: From                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Met Ala Cys Asn Cys Gln Leu Met Gln Asp Th - #r Pro Leu Leu Lys Phe         25                                                                            - Pro Cys Pro Arg Leu Ile Leu Leu Phe Val Le - #u Leu Ile Arg Leu Ser         10                                                                            - Gln Val Ser Ser Asp Val Asp Glu Gln Leu Se - #r Lys Ser Val Lys Asp         #                10                                                           - Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pr - #o His Glu Asp Glu Ser         #             25                                                              - Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Ly - #s Val Val Leu Ser Val         #         40                                                                  - Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Ty - #r Lys Asn Arg Thr Leu         #     55                                                                      - Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Le - #u Gly Leu Val Leu Ser         # 75                                                                          - Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Ly - #s Lys Glu Arg Gly Thr         #  90                                                                         - Tyr Gly Val Lys His Leu Ala Leu Val Lys Le - #u Ser Ile Lys Ala Asp         #            105                                                              - Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly As - #n Pro Ser Ala Asp Thr         #       120                                                                   - Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Ph - #e Pro Lys Pro Arg Phe         #   135                                                                       - Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gl - #y Ile Asn Thr Thr Ile         140                 1 - #45                 1 - #50                 1 -       #55                                                                           - Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Il - #e Ser Ser Gln Leu Asp         #               170                                                           - Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cy - #s Leu Ile Lys Tyr Gly         #           185                                                               - Asp Ala His Val Ser Glu Asp Phe Thr Trp Gl - #u Lys Pro Pro Glu Asp         #       200                                                                   - Pro Pro Asp Ser Lys Asn Thr Leu Val Leu Ph - #e Gly Ala Gly Phe Gly         #   215                                                                       - Ala Val Ile Thr Val Val Val Ile Val Val Il - #e Ile Lys Cys Phe Cys         220                 2 - #25                 2 - #30                 2 -       #35                                                                           - Lys His Arg Ser Cys Phe Arg Arg Asn Glu Al - #a Ser Arg Glu Thr Asn         #               250                                                           - Asn Ser Leu Thr Phe Gly Pro Glu Glu Ala Le - #u Ala Glu Gln Thr Val         #           265                                                               - Phe Leu                                                                     - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #    39            CTTG AGATCACAGT TCTCTCTAC                                  - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #      37          GAGC GGAGTGGACA CCTGTGG                                    - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                # 20               AAGC                                                       - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #           30     GGAG GCTGAGGTCC                                            - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 78 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - GCATTTTAAG CTTTTTCCTG ATCAGGAGCC CAAATCTTCT GACAAAACTC AC - #ACATCTCC         60                                                                          #  78              CC                                                         - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                # 20               AGGG                                                       - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - GAGCATTTTC CTGATCAGGA GTCCAAATAT GGTCCCCCAT CCCATCATCC CC - #AGGTAAGC         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                - GCAGAGGAAT CGAGCTCGGT ACCCGGGGAT CCCCAGTGTG GGGACAGTGG GA - #CCGCTCTG         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 59 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                - GGGTTTTGGG GGGAAGAGGA AGACTGACGG TGCCCCCTCG GCTTCAGGTG CT - #GAGGAAG          59                                                                          - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 56 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                - CATCTCTTCC TCAGCACCTG AAGCCGAGGG GGCACCGTCA GTCTTCCTCT TC - #CCCC             56                                                                          - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 99 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                - CGCACGTGAC CTCAGGGGTC CGGGAGATCA TGAGAGTGTC CTTGGGTTTT GG - #GGGGAACA         60                                                                          #    99            CCCC TCGAACTCAG GTGCTGAGG                                  - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 98 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                - CCTCAGCACC TGAGTTCGAG GGGGCACCAT CAGTCTCCTG TTCCCCCCAA AA - #CCCAAGGA         60                                                                          #     98           CGGA CCCCTGAGGT CACGTGCG                                   - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 330 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..330                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                - GCT CCT CTG AAG ATT CAA GCT TAT TTC AAT GA - #G ACT GCA GAC CTG CCA           48                                                                          Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Gl - #u Thr Ala Asp Leu Pro           #                 15                                                          - TGC CAA TTT GCA AAC TCT CAA AAC CAA AGC CT - #G AGT GAG CTA GTA GTA           96                                                                          Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Le - #u Ser Glu Leu Val Val           #             30                                                              - TTT TGG CAG GAC CAG GAA AAC TTG GTT CTG AA - #T GAG GTA TAC TTA GGC          144                                                                          Phe Trp Gln Asp Gln Glu Asn Leu Val Leu As - #n Glu Val Tyr Leu Gly           #         45                                                                  - AAA GAG AAA TTT GAC AGT GTT CAT TCC AAG TA - #T ATG GGC CGC ACA AGT          192                                                                          Lys Glu Lys Phe Asp Ser Val His Ser Lys Ty - #r Met Gly Arg Thr Ser           #     60                                                                      - TTT GAT TCG GAC AGT TGG ACC CTG AGA CTT CA - #C AAT CTT CAG ATC AAG          240                                                                          Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu Hi - #s Asn Leu Gln Ile Lys           # 80                                                                          - GAC AAG GGC TTG TAT CAA TGT ATC ATC CAT CA - #C AAA AAG CCC ACA GGA          288                                                                          Asp Lys Gly Leu Tyr Gln Cys Ile Ile His Hi - #s Lys Lys Pro Thr Gly           #                 95                                                          - ATG ATT CGC ATC CAC CAG ATG AAT TCT AGG CT - #G TCA GTG CTT                 # 330                                                                         Met Ile Arg Ile His Gln Met Asn Ser Arg Le - #u Ser Val Leu                   #           110                                                               - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 110 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                - Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Gl - #u Thr Ala Asp Leu Pro         #                 15                                                          - Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Le - #u Ser Glu Leu Val Val         #             30                                                              - Phe Trp Gln Asp Gln Glu Asn Leu Val Leu As - #n Glu Val Tyr Leu Gly         #         45                                                                  - Lys Glu Lys Phe Asp Ser Val His Ser Lys Ty - #r Met Gly Arg Thr Ser         #     60                                                                      - Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu Hi - #s Asn Leu Gln Ile Lys         # 80                                                                          - Asp Lys Gly Leu Tyr Gln Cys Ile Ile His Hi - #s Lys Lys Pro Thr Gly         #                 95                                                          - Met Ile Arg Ile His Gln Met Asn Ser Arg Le - #u Ser Val Leu                 #           110                                                               - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 306 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..310                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                - GCT AAC TTC AGT CAA CCT GAA ATA GTA CCA AT - #T TCT AAT ATA ACA GAA           48                                                                          Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Il - #e Ser Asn Ile Thr Glu           #                 15                                                          - AAT GTG TAC ATA AAT TTG ACC TGC TCA TCT AT - #A CAC GGT TAC CCA GAA           96                                                                          Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Il - #e His Gly Tyr Pro Glu           #             30                                                              - CCT AAG AAG ATG AGT GTT TTG CTA AGA ACC AA - #G AAT TCA ACT ATC GAG          144                                                                          Pro Lys Lys Met Ser Val Leu Leu Arg Thr Ly - #s Asn Ser Thr Ile Glu           #         45                                                                  - TAT GAT GGT ATT ATG CAG AAA TCT CAA GAT AA - #T GTC ACA GAA CTG TAC          192                                                                          Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp As - #n Val Thr Glu Leu Tyr           #     60                                                                      - GAC GTT TCC ATC AGC TTG TCT GTT TCA TTC CC - #T GAT GTT ACG AGC AAT          240                                                                          Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pr - #o Asp Val Thr Ser Asn           # 80                                                                          - ATG ACC ATC TTC TGT ATT CTG GAA ACT GAC AA - #G ACG CGG CTT TTA TCT          288                                                                          Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Ly - #s Thr Arg Leu Leu Ser           #                 95                                                          # 306              TA GAG                                                     Ser Pro Phe Ser Ile Glu                                                                   100                                                               - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 102 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                - Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Il - #e Ser Asn Ile Thr Glu         #                 15                                                          - Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Il - #e His Gly Tyr Pro Glu         #             30                                                              - Pro Lys Lys Met Ser Val Leu Leu Arg Thr Ly - #s Asn Ser Thr Ile Glu         #         45                                                                  - Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp As - #n Val Thr Glu Leu Tyr         #     60                                                                      - Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pr - #o Asp Val Thr Ser Asn         # 80                                                                          - Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Ly - #s Thr Arg Leu Leu Ser         #                 95                                                          - Ser Pro Phe Ser Ile Glu                                                                 100                                                               - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #                47CACC ATGGGGGTAC TGCTCACACA GAGGACG                         - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 63 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                - AGTCTCATTG AAATAAGCTT GAATCTTCAG AGGAGCCATG CTGGCCATGC TT - #GGAAACAG         60                                                                          #             63                                                              - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 62 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                - CTCCTGTTTC CAAGCATGGC CAGCATGGCT CCTCTGAAGA TTCAGGCTTA TT - #TCAATGAG         60                                                                          #              62                                                             - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                #             51CTCATTA CTGATCAAGC ACTGACAGTT CAGAATTCAT C                    - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 63 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                - AGAAATTGGT ACTATTTCAG GTTGACTGAA GTTAGCCATG CTGGCCATGC TT - #GGAAACAG         60                                                                          #             63                                                              - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 62 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                - CTCCTGTTTC CAAGCATGGC CAGCATGGCT AACTTCAGTC AACCTGAAAT AG - #TACCAATT         60                                                                          #              62                                                             - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 105 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                - CTCAAGCTTG CCACCATGGG GGTACTGCTC ACACAGAGGA CGCTGCTCAG TC - #TGGTCCTT         60                                                                          #                 105AT GGCGAGCATG GGTCTTTCTC ACTTC                           - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: oligonucleotide                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #45                ATTA CTGATCAGGA AAATGCTCTT GCTTG                           __________________________________________________________________________

We claim:
 1. An isolated peptide that costimulates T cell proliferationor T cell cytokine production and that binds CD28 or CTLA4, said peptideencoded by a nucleic acid molecule which hybridizes in 6.0×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash inabout 0.2×SSC at a temperature of about 50° C. to a nucleic acidmolecule which encodes a human B7-2 peptide comprising the amino acidsequence shown in FIG. 8 (SEQ ID NO:2).
 2. An isolated peptide thatcostimulates T cell proliferation or T cell cytokine production and thatbinds CD28 or CTLA4, said peptide encoded by a nucleic acid moleculewhich hybridizes in 6.0×sodium chloride/sodium citrate (SSC) at about45° C., followed by a wash in about 0.2×SSC at a temperature of about50° C. to a nucleic acid molecule which encodes a mouse B7-2 peptidecomprising the amino acid sequence shown in FIG. 14 (SEQ ID NO:23). 3.An isolated peptide consisting of amino acid residues 24-245 of theamino acid sequence of the human B7-2 peptide shown in FIG. 8 (SEQ IDNO:2).
 4. An isolated peptide consisting of the amino acid sequence ofthe mouse B7-2 peptide shown in FIG. 14 (SEQ ID NO:23).
 5. An isolatedpeptide consisting of the extracellular domain of the amino acidsequence shown in FIG. 14 (SEQ ID NO:23).
 6. The isolated peptide ofclaim 5, wherein the extracellular domain consists of amino acidresidues 24-246 of the amino acid sequence shown in FIG. 14 (SEQ IDNO:23).
 7. A peptide produced by recombinant expression of a DNAcomprising the nucleotide sequence shown in FIG. 8 (SEQ ID NO:1).
 8. Apeptide produced by recombinant expression of a DNA comprising thecoding region of the nucleotide sequence shown in FIG. 8 (SEQ ID NO:1).